Detecting neoplasm

ABSTRACT

This document relates to methods and materials for detecting premalignant and malignant neoplasms. For example, methods and materials for determining whether or not a stool sample from a mammal contains nucleic acid markers or polypeptide markers of a neoplasm are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/197,105, filed Jun. 29, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/827,013, now U.S. Pat. No. 9,309.800, which is acontinuation of U.S. patent application Ser. No. 14/168,552, now U.S.Pat. No. 9,121,070, which is a continuation of U.S. patent applicationSer. No. 12/866,558, now U.S. Pat. No. 8,673,555, which is a Section 371U.S. national stage entry of international Patent Application No.PCT/US2009/033793, filed Feb. 11, 2009, which claims priority to expiredU.S. Provisional Patent Application No. 61/029,221, filed Feb. 15, 2008,the contents of which are hereby incorporated by reference in theirentireties.

BACKGROUND 1. Technical Field

This document relates to methods and materials involved in detectingpremalignant and malignant neoplasms (e.g., colorectal and pancreaticcancer).

2. Background Information

About half of all cancer deaths in the United States result fromaero-digestive cancer. For example, of the estimated annual cancerdeaths, about 25 percent result from lung cancer; about 10 percentresult from colorectal cancer; about 6 percent result from pancreascancer; about 3 percent result from stomach cancer; and about 3 percentresult from esophagus cancer. In addition, over 7 percent of the annualcancer deaths result from other aero-digestive cancers such asnaso-oro-pharyngeal, bile duct, gall bladder, and small bowel cancers.

SUMMARY

This document relates to methods and materials for detectingpremalignant and malignant neoplasms (e.g., colorectal and pancreaticcancer). For example, this document provides methods and materials thatcan be used to determine whether a sample (e.g., a stool sample) from amammal contains a marker for a premalignant and malignant neoplasm suchas a marker from a colonic or supracolonic aero-digestive neoplasmlocated in the mammal. The detection of such a marker in a sample from amammal can allow a clinician to diagnose cancer at an early stage. Inaddition, the analysis of a sample such as a stool sample can be muchless invasive than other types of diagnostic techniques such asendoscopy.

This document is based, in part, on the discovery of particular nucleicacid markers, polypeptide markers, and combinations of markers presentin a biological sample (e.g., a stool sample) that can be used to detecta neoplasm located, for example, in a mammal's small intestine, gallbladder, bile duct, pancreas, liver, stomach, esophagus, lung, ornaso-oro-pharyngeal airways. For example, as described herein, stool canbe analyzed to identify mammals having cancer. Once a particular mammalis determined to have stool containing a neoplasm-specific marker orcollection of markers, additional cancer screening techniques can beused to identify the location and nature of the neoplasm. For example, astool sample can be analyzed to determine that the patient has aneoplasm, while magnetic resonance imaging (MRI), endoscopic analysis,and tissue biopsy techniques can be used to identify the location andnature of the neoplasm. In some cases, a combination of markers can beused to identify the location and nature of the neoplasm withoutadditional cancer screening techniques such as MRI, endoscopic analysis,and tissue biopsy techniques.

In general, one aspect of this document features a method of detectingpancreatic cancer in a mammal. The method comprises, or consistsessentially of determining the ratio of an elastase 3A polypeptide to apancreatic alpha-amylase polypeptide present within a stool sample. Thepresence of a ratio greater than about 0.5 indicates that the mammal haspancreatic cancer. The presence of a ratio less than about 0.5 indicatesthat the mammal does not have pancreatic cancer.

In another aspect, this document features a method of detectingpancreatic cancer in a mammal. The method comprises or consistsessentially of determining the level of an elastase 3A polypeptide in astool sample from the mammal. The presence of an increased level of anelastase 3A polypeptide, when compared to a normal control level, isindicative of pancreatic cancer in the mammal.

In another aspect, this document features a method of detectingpancreatic cancer in a mammal. The method comprises, or consistsessentially of, determining the level of a carboxypeptidase Bpolypeptide in a stool sample from the mammal. An increase in the levelof a carboxypeptidase B polypeptide, when compared to a normal controllevel, is indicative of pancreatic cancer in the mammal.

In another aspect, this document features a method of detectingpancreatic cancer in a. mammal. The method comprises, or consistsessentially of, determining whether or not a stool sample from themammal comprises a ratio of a carboxypeptidase B polypeptide to acarboxypeptidase A2 polypeptide that is greater than about 0.5. Thepresence of the ratio greater than about 0.5 indicates that the mammalhas pancreatic cancer.

In another aspect, this document features a method of detecting canceror pre-cancer in a mammal. The method comprises, or consists essentiallyof, determining whether or not a stool sample from the mammal has anincrease in the number of DNA fragments less than 200 base pairs inlength, as compared to a normal control. The presence of the increase inthe number of DNA fragments less than 200 base pairs in length indicatesthat the mammal has cancer or pre-cancer. The DNA fragments can be lessthan 70 base pairs in length.

In another aspect, this document features a method of detectingcolorectal cancer or pre-cancer in a mammal. The method comprises, orconsists essentially of, determining whether or not a stool sample fromthe mammal has an elevated K-ras (Kirsten rat sarcoma-2 viral(v-Ki-ras2) oncogene homolog (GenBank accession no. NM_033360;gi|34485724|)) mutation score, an elevated BMP3 (hone morphogeneticprotein 3 (GenBank accession no. M22491; gi|179505)) methylation status,and an elevated level of human DNA as compared to a normal control. Thepresence of the elevated K-ras mutation score, elevated BMP3 methylationstatus, and elevated level of human DNA level indicates that the mammalhas colorectal cancer or pre-cancer. The K-ras mutation score can bemeasured by digital melt curve analysts. The K-ras mutation score can bemeasured by quantitative allele specific PCR.

In another aspect, this document features a method of detectingaero-digestive cancer or pre-cancer in a mammal. The method comprises,or consists essentially of, determining whether or not a stool samplefrom the mammal has an elevated K-ras mutation score, an elevated BMP3methylation status, and an elevated level 0 f human DNA as compared to anormal control. The presence of the elevated K-ras mutation score,elevated BMP3 methylation status, and elevated level of human DNA levelindicates that the mammal has aero-digestive cancer or pre-cancer. TheK-ras mutation score can be measured b digital melt curve analysis. TheK-ras mutation score can be measured by quantitative allele-specificPCR. The method can further comprise determining whether or not a stoolsample from the mammal has an elevated APC mutation score. The APCmutation score can be measured by digital melt curve analysis.

In another aspect, this document features a method of detectingaero-digestive cancer or pre-cancer in a mammal. The method comprises,or consists essentially of, determining whether or not the mammal has atleast one mutation in six nucleic acids selected from the groupconsisting of p16, p53, k-ras, APC (adenomatosis polyposis coli tumorsuppressor (GenBank accession no. NM_000038; gi|189011564)), SMAD4 (SMADfamily member 4 (GenBank accession no. NM_005359; gi|195963400)), EGFR(epidermal growth factor receptor (GenBank accession no. NM_005228gi|41327737)), CTNNBI1 (catenin (cadherin-associated protein), beta 1(88 kD) (GenBank accession no. X87838; gi|1154853 and BRAF (B-Rafproto-oncogene serine/threonine-protein kinase (p94) (GenBank accessionno. NM_004333; gi|187608632)) nucleic acids. The presence of at leastone mutation in each of the six nucleic acids indicates that the mammalhas aero-digestive cancer or pre-cancer. The method can further comprisedetermining whether or not a stool sample from the mammal has anelevated level of a carboxypeptidase B polypeptide as compared to anormal control. The presence of the elevated level of a caboxypeptidaseB polypeptide indicates that the mammal has aerodigestive cancer orpre-cancer in the mammal. The method can further comprise determiningwhether or not a stool sample from the mammal has an elevated amount ofDNA fragments less than 70 base pairs in length as compared to a normalcontrol. The presence of the elevated amount of DNA fragments less than70 base pairs in length indicates that the mammal has aero-digestivecancer or pre-cancer. The method can further comprise determiningwhether or not a stool sample from the mammal has an elevated amount ofDNA fragments greater than 100 base pairs in length as compared tonormal controls. The presence of the elevated amount of DNA fragmentsgreater than 100 base pairs in length indicates that the mammal hasaero-digestive cancer or pre-cancer. The method can further comprisedetermining whether or not a stool sample from the mammal has anelevated BMP3 methylation status. The elevated BMP3 methylation statuslevel indicates that the mammal has aero-digestive cancer or pre-cancer.The determining step can comprise using digital melt curve analysis.

In another aspect, this document features a method of detectingaero-digestive cancer or pre-cancer in a mammal. The method comprises,or consists essentially of, measuring mutations in a matrix marker panelin a stool sample. The marker panel can comprise measuring DNA mutationsin p16, p53, k-ras, APC, SMAD4, EGER, CTNNB1. and BRAF nucleic acids.The presence of a mutation in each of the nucleic acids is indicative ofthe presence of aero-digestive cancer or pre-cancer in a mammal.

In another aspect, this document features a method of detectingaero-digestive cancer in a mammal. The method comprises, or consistsessentially of, determining whether or not the methylation status of anALX4 (aristaless-like homeobox 4 (GenBank accession no. AF294629,gi|10863748|)) nucleic acid in a stool sample from the mammal iselevated, as compared to a normal control. The presence of an elevatedALX4 methylation status indicates the presence of aero-digestive cancerin the mammal.

In another aspect, this document features a method of diagnosingpancreatic cancer in a mammal. The method comprises, or consistsessentially of, obtaining a stool sample from the mammal, determiningthe ratio of an elastase 3A polypeptide to a pancreatic alpha-amylasepolypeptide present within a stool sample, and communicating a diagnosisof pancreatic cancer if the ratio is greater than about 0.5, therebydiagnosing the mammal with pancreatic cancer.

In another aspect, this document features a method of diagnosing amammal with pancreatic cancer. The method comprises, or consistsessentially of, obtaining a stool sample from the mammal, measuringmutations in a matrix marker panel of nucleic acids present in thesample, determining the ratio of a carboxypeptidase B polypeptide to acarboxypeptidase A2 polypeptide present within the sample, andcommunicating a diagnosis of pancreatic cancer or pre-cancer if amutation is detected in each of the marker panel nucleic acids and theratio is greater than 0.5, thereby diagnosing the mammal. The matrixmarker panel comprises or consists essentially of p16, p53, k-ras, APC,SMAD4, EGFR, CTNNB1, and BRAE nucleic acids.

In another aspect, this document features method of diagnosing a mammalwith colorectal cancer. The method comprises, or consists essentiallyof, obtaining a stool sample from the mammal, detecting mutations in amatrix marker panel comprising of p16, p53, k-ras, APC, SMAD4, EGFR,CTNNB1, and BRAF nucleic acids in DNA present within the sample,measuring the level of a serotransferrin polypeptide present within thesample, and communicating a diagnosis of colorectal cancer or pre-cancerif a mutation is detected in each of the nucleic acids and the level ofa serotransferrin polypeptide is elevated as compared to a referencelevel, thereby diagnosing the mammal.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Adjusted Cut-off Levels with Quantitative Stool Markers toAchieve 95% Specificity across Age and Gender using the Q-LEAD Model.Solid line for women, dotted line for men.

FIG. 2: Sensitive and specific detection of pancreatic cancer by fecalratio of carboxypeptidase B: carboxypeptidase A2. Note that ratio instools from patients with colorectal cancer is no different from ratioswith healthy controls.

FIG. 3: Elastase levels quantified in stools from patients withpancreatic cancer, patients with colorectal cancer, and healthycontrols.

FIG. 4: Ratio of elastase 3A:pancreatic alpha amylase differentiatespatients with pancreatic cancer from patients with colorectal cancer andfrom healthy controls.

FIG. 5: Digital Melt Curve to detect mutations by targeted gene scanning(temperature (x-axis) v. temperature-normalized fluorescence (y-axis)).Eight pairs of primers, which amplify 100-350 bp gene fragments, wereused to scan K-ras and APC genes and detect mutations (substitution anddeletion mutations, respectively) at 1% mutant/wild type ratio.

FIG. 6: Quantitive detection of low abundance mutations by Digital MeltCurve Assay. Varying Mutant: Wild-type ratios of K-r-as and APC genemixtures were prepared and assayed blindly by Digital Melt Curve.

FIG. 7: High analytical sensitivity ^(1w) Digital Melt Curve(temperature (x-axis) v. temperature-normalized fluorescence (y-axis)).To test the detection limit of digital melt curve (DMC) assay, mutantcopies were spiked in wild-type copies at 0.1. 0.5, 1, 5, and 10%dilutions. DMC could detect up to 0.1% mutant/wild-type level when 1000copies were dispersed to one 96-well plate. The numbers of positivewells increased proportionally when spiked mutant copies were increased.A pair of primers that amplify 248 bp K-ras gene fragment were used asan example here. Primers that amplify-119 bp K-ras gene, 162 bp APCgene, and 346 bp APC genes were also used to test the detection limitand quantitative property of DMC.

FIG. 8: Superior screen detection of colorectal precancerous polyps byDigital Melt Curve (DMC). Histogram compares sensitivity by DMC withthat by common fecal occult blood tests (Hemoccult and HemoccultSENSA)and by the commercial stool DNA test (PreGenPlus, Exact Sciences).Detection by DMC was significantly better than by any other test(p<0.05).

FIG. 9: Distributions of short fragment human DNA (short DNA) and longfragment human DNA (long DNA) in stools from patients with normalcolonoscopy, large precancerous adenomas, and colorectal cancer. HumanDNA quantified by an assay of Alu repeats. Short DNA represents 45 bpfragment amplification products, and long DNA represents 245 bpamplification products.

FIG. 10: Stool distributions of short and long DNA in patients withpancreatic cancer and in healthy controls. Short DNA represents 45 bpfragment amplification products, and long DNA represents 245 bpamplification products.

FIG. 11: Methylated BMP3 gene in stool for detection of colorectalneoplasia. Methylated BMP3 was blindly quantified in stools frompatients with colorectal cancers, precancerous adenomas, and normalindividuals with real-time methylation-specific PCR. Each circlerepresents a stool sample.

FIG. 12: Frequency of Specific Base Changes in Colorectal Tumors.

DETAILED DESCRIPTION

This document provides methods and materials related to detecting aneoplasm in a mammal (e.g., a human). For example, this documentprovides methods and materials for using nucleic acid markers,polypeptide markers, and combinations of markers present in a biologicalsample (e.g., a stool sample) to detect a neoplasm in a mammal. Such aneoplasm can be a cancer or precancer in the head and neck, lungs andairways, esophagus, stomach, pancreas, bile ducts, small bowel, orcolorectum. It will be appreciated that the methods and materialsprovided herein can be used to detect neoplasm markers in a mammalhaving a combination of different neoplasms. For example, the methodsand materials provided herein can be used to detect nucleic acid andpolypeptide markers in a human having lung and stomach neoplasms.

In some cases, the methods and materials provided herein can he used toquantify multiple markers in biological samples, stool) to yield highsensitivity for detection of lesions (e.g., neoplasms), while preservinghigh specificity. Such methods can include, for example, a logisticmodel that adjusts specificity cut-offs based on age, gender, or othervariables in a target population to be tested or screened.

In some cases, the methods and materials provided herein can he used todetermine whether a mammal (e.g., a human) has colorectal cancer orpancreatic cancer. For example, serotransferin, methylated BMP3, andmutant BRAF markers in stool can be used to identify a mammal as likelyhaving colorectal cancer, while mutant p16, carboxypeptidase B/A, andelastase 2A markers can be used to identify a mammal as likely havingpancreatic cancer.

Any suitable method can be used to detect a nucleic acid marker in amammalian stool sample. For example, such methods can involve isolatingDNA from a stool sample, separating out one or more particular DNAs fromthe total DNA, subjecting the DNAs to bisulfite treatment, anddetermining whether the separated DNAs are abnormally methylated (e.g.,hypermethylated or hypomethylated). In some cases, such methods caninvolve isolating DNA from a stool sample and determining the presenceor absence of DNA having a particular size (e.g., short DNA). It isnoted that a single stool sample can be analyzed for one nucleic acidmarker or for multiple nucleic acid markers. For example, a stool samplecan be analyzed using assays that detect a panel of different nucleicacid markers. In addition, multiple stool samples can be collected froma single mammal and analyzed as described herein.

Nucleic acid can be isolated from a stool sample using, for example, akit such as the QIAamp DNA Stool Mini Kit (Qiagen Inc., Valencia,Calif.). In addition, nucleic acid can be isolated from a stool sampleusing the following procedure: (1) homogenizing samples in an excessvolume (>1:7 w:v) of a stool stability buffer (0.5M Tris pH 9.0, 150 mMEDTA, 10 mM NaCl) by shaking or mechanical mixing; (2) centrifuging a 10gram stool equivalent of each sample to remove all particulate matter;(3) adding 1 of 100 μg/μL, RNase A to the supernatant and incubating at37° C. for 1 hour; (4) precipitating total nucleic acid with 1/10 volume3M NaAc and an equal volume isopropanol; and (5) centrifuging and thenresuspending the DNA pellet in TB (0.01 M Tris pH 7.4, 0.001 NI EDTA).U.S. Pat. Nos. 5,670,325; 5,741,650, 5,928,870; 5,952,178, and 6,020,137also describe various methods that can be used to prepare and analyzestool samples.

One or more specific nucleic acid fragments can be purified from anucleic acid preparation using, for example, a modifiedsequence-specific hybrid capture technique (see. e.g., Ahlquist et al.(2000) Gastroenterology, 119:1219-1227). Such a protocol can involve:(1) adding 300 μL of sample preparation to an equal volume of a 6 Mguanidine isothiocyanate solution containing 20 pmol biotinylatedoligonucleotides (obtained from, for example, Midland Certified ReagentCo., Midland, Tex.) with sequences specific for the DNA fragments to beanalyzed; (2) incubating for two hours at 25° C.; (3) addingstreptavidin coated magnetic beads to the solution and incubating for anadditional hour at room temperature; (4) washing the bead/hybrid capturecomplexes four times with IX B+W buffer (I M NaCl, 0.01 M Tris-HCl pH7.2, 0.001 M EDTA, 0.1% Tweet) 20); and (5) eluting the sequencespecific captured DNA into 35 μL L-TE (1 mM Tris pH 7.4, 0.1 M EDTA) byheat denaturation of the bead/hybrid capture complexes. Any othersuitable technique also can be used to isolate specific nucleic acidfragments.

Nucleic acid can be subjected to bisulfite treatment to convertunmethylated cytosine residues to uracil residues, while leaving any5-methylcytosine residues unchanged. A bisulfite reaction can beperformed using, for example, standard techniques: (1) denaturingapproximately 1 μg of genomic DNA (the amount of DNA can be less whenusing micro-dissected DNA specimens) for 15 minutes at 45° C. with 2 NNaOH; (2) incubating with 0.1 M hydroquinone and 3.6 M sodium bisulfite(pH 5.0) at 55° C. for 4-12 hours; (3) purifying the DNA from thereaction mixture using standard (e.g., commercially-available) DNAminiprep colunms or other standard techniques for DNA purification; (4)resuspending the purified DNA sample in 55 μL water and adding 5 μl 3 NMOH for a desulfonation reaction that typically is performed at 40° C.for 5-10 minutes; (5) precipitating the DNA sample with ethanol, washingthe DNA, and resuspending the DNA in an appropriate volume of water.Bisulfite conversion of cytosine residues to uracil also can be achievedusing other methods (e.g., the CpGenome™ DNA Modification Kit fromSerologicals Corp., Norcross, Ga.).

Any appropriate method can be used to determine whether a particular DNAis hypermethylated or hypomethylated. Standard PCR techniques, forexample, can be used to determine which residues are methylated, sinceunmethylated cytosines converted to uracil are replaced by thymidineresidues during PCR. PCR reactions can contain, for example, 10 μL ofcaptured DNA that either has or has not been treated with sodiumbisulfite, IX PCR buffer, 0.2 mM dNTPs, 0.5 μM sequence specific primers(e.g., primers flanking a CpG island within the captured DNA), and 5units DNA polymerase (e.g., Amplitaq DNA polymerase from PE AppliedBiosystems, Norwalk, Conn.) in a total volume of 50 μl. A typical PCRprotocol can include, for example, an initial denaturation step at 94°C. for 5 min, 40 amplification cycles consisting of 1 minute at 94° C.,1 minute at 60° C., and 1 minute at 72° C., and a final extension stepat 72° C. for 5 minutes.

To analyze which residues within a captured DNA are methylated, thesequences of PCR products corresponding to samples treated with andwithout sodium bisulfite can be compared. The sequence from theuntreated DNA will reveal the positions of all cytosine residues withinthe PCR product. Cytosines that were methylated will be converted tothymidine residues in the sequence of the bisulfite-treated DNA, whileresidues that were not methylated will be unaffected by bisulfatetreatment.

Purified nucleic acid fragments from a stool sample or samples can beanalyzed to determine the presence or absence of one or more somaticmutations. Mutations can be single base changes, shortinsertion/deletions, or combinations thereof Methods of analysis caninclude conventional Sanger based sequencing, pyrosequencing, nextgeneration sequencing, single molecule sequencing, and sequencing bysynthesis. In some cases, mutational status can be determined by digitalPCR followed by high resolution melting curve analysis. In other cases,allele specific primers or probes in conjunction with amplificationmethods can be used to detect specific mutations in stool DNA. Themutational signature can comprise not only the event of a base orsequence change in a specific gene, but also the location of the changewithin the gene, whether it is coding, non-coding, synonymous ornon-synonymous, a transversion or transition, and the dinucleotidesequence upstream and downstream from the alteration.

In some cases, a sample can be assessed for the presence or absence of apolypeptide marker. For example, any appropriate method can be used toassess a stool sample for a polypeptide marker indicative of a neoplasm.For example, a stool sample can be used in assays designed to detect oneor more polypeptide markers. Appropriate methods such as those describedelsewhere (Aebersold and Mann, Nature, 422:198-207 (2003) and McDonaldand Yates, Dis. Markers, 18:99-105 (2002)) can be adapted or designed todetect polypeptides in a stool. For example, single-reaction monitoringusing a TSQ mass spectrometer can specifically target polypeptides in astool sample. High resolution instruments like the LTQ-FT or LTQorbitrap can be used to detect polypeptides present in a stool sample.

The term “increased level” as used herein with respect to the level ofan elastase 3A polypeptide is any level that is above a median elastase3A polypeptide level in a stool sample from a random population ofmammals (e.g., a random population of 10, 20, 30, 40, 50, 100, or 500mammals) that do not have an aero-digestive cancer. Elevated polypeptidelevels of an elastase 3A polypeptide can be any level provided that thelevel is greater than a corresponding reference level. For example, anelevated level of an elastase 3A polypeptide can be 0.5, 1, 2, 3, 4. 5,6 7, 8, 9, 10, or more fold greater than the reference level of elastase3A polypeptide in a normal sample. It is noted that a reference levelcan he any amount. For example, a reference level for an elastase 3Apolypeptide can he zero. In some cases, an increased level of anelastase 3A polypeptide can be any detectable level of an elastase 3Apolypeptide in a stool sample.

The term “increased level” as used herein with respect to the level ofan carboxypeptidase B polypeptide level is any level that is above amedian carboxypeptidase B poly peptide level in a stool sample from arandom population of mammals (e.g., a random population of 10, 20, 30,40, 50, 100, or 500 mammals) that do not have an aero-digestive cancer.Elevated polypeptide levels of carboxypeptidase B polypeptide can be anylevel provided that the level is greater than a corresponding referencelevel. For example, an elevated level of carboxypeptidase B polypeptidecan be 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold greater than thereference level carboxypeptidase B polypeptide observed in a normalstool sample. It is noted that a reference level can be any amount. Forexample, a reference level for a carboxypeptidase B polypeptide can bezero. In some cases, an increased level of a carboxypeptidase Bpolypeptide can be any detectable level of a carboxypeptidase Bpolypeptide in a stool sample.

The term “increased level” as used herein with respect to the level ofDNA fragments less than about 200 or less than about 70 base pairs inlength is any level that is above a median level of DNA fragments, lessthan about 200 or less than about 70 base pairs in length in a stoolsample from a random population of mammals (e.g., a random population of10, 20, 30, 40, 50, 100, or 500 mammals) that do not have anaero-digestive cancer. In some cases, an increased level of DNAfragments less than about 200 or less than about 70 base pairs in lengthcan be any detectable level of DNA fragments less than about 200 or lessthan about 70 base pairs in length in a stool sample.

The term “elevated methylation” as used herein with respect to themethylation status of a BMP3 or ALX nucleic acid is any methylationlevel that is above a median methylation level in a stool sample from arandom population of mammals (e.g., a random population of 10, 20, 30,40, 50. 100, or 500 mammals) that do not have an aero-digestive cancer.Elevated levels of BMP3 or ALX methylation can be any level providedthat the level is greater than a corresponding reference level. Forexample, an elevated level of BMP3 or ALX methylation can be 0.5, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more fold greater than the reference levelmethylation observed in a normal stool sample. It is noted that areference level can be any amount.

The term “elevated mutation score” as used herein with respect todetected mutations in a matrix panel of particular nucleic acid markersis any mutation score that is above a median mutation score in a stoolsample from a random population of mammals (e.g., a random population of10, 20, 30, 40, 50, 100, or 500 mammals) that do not have anaero-digestive cancer. An elevated mutation score in a matrix panel ofparticular nucleic acid markers can be any score provided that the scoreis greater than a corresponding reference score. For example, anelevated score of K-ras or APC mutations can be 0.5, 1, 2, 3, 4. 5, 6,7, 8, 9, 10, or more fold greater than the reference score of K-ras orAPC mutations observed in a normal stool sample it is noted that areference score can be any amount.

In some cases, a ratio of particular polypeptide markers can bedetermined and used to identify a mammal having an aero-digestive cancer(e.g., a colorectal cancer or a pancreatic cancer). For example, a ratioprovided herein (e.g., the ratio of carboxypeptidase B polypeptidelevels to carboxypeptidase A2 polypeptide levels) to can be used asdescribed herein to identify a mammal having a particular neoplasm(e.g., pancreatic cancer).

In some cases, a matrix marker panel can be used to identify mammalshaving an aero-digestive cancer (e.g., a colorectal cancer or apancreatic cancer). In some cases, such panel also can identify thelocation of the aero-digestive cancer. Such a panel can include nucleicacid markers, polypeptide markers, and combinations thereof and canprovide information about a mutated marker gene, the mutated region ofthe marker gene, and/or type of mutation. For example, data can heanalyzed using a statistical model to predict tumor site (e.g.,anatomical location or tissue of origin) based on inputs from sequencingdata (such as by specific nucleic acid or combination of nucleic acidsmutated, specific mutational location on a nucleic acid, and nature ofmutation insertion, deletion, transition, or transversion) or by anycombination thereof) and/or data from polypeptide or other types ofmarkers. For example, a Site of Tumor Estimate (SITE) model can be usedto predict tumor site using a matrix panel of markers that are presentto variable extent across tumors.

In some cases, data can be analyzed using quantified markers to create alogistic model, which can have both high sensitivity and highspecificity. For example, a logistic model can also incorporatepopulation variables like gender and age to adjust cut-off levels fortest positivity and thereby optimize assay performance in a screeningsetting. In some cases, a Quantitative Logistic to Enhance AccurateDetection (Q-LEAD) Model can be used with any marker class orcombination of markers as long as they can be quantified.

This document also provides methods and materials to assist medical orresearch professionals in determining whether or not a mammal has anaero-digestive cancer. Medical professionals can be, for example,doctors, nurses, medical laboratory technologists, and pharmacists.Research professionals can be, for example, principle investigators,research technicians, postdoctoral trainees, and graduate students. Aprofessional can be assisted by (1) determining the ratio of particularpolypeptide markers in a stool sample, and (2) communicating informationabout the ratio to that professional, for example. In some cases, aprofessional can be assisted by (1) determining the level of human DNA,the methylation status of genes such as BMP3, and the mutation score ofgenes such as APC and K-ras, and (2) communicating information about thelevel of DNA, the methylation status of particular genes, and themutation score of particular genes to the professional in some cases, aprofessional can be assisted by (1) detecting mutations incancer-related genes such as K-ras, p53, APC, p16, EGFR, BRAF, andSMAD4, as a matrix marker panel, and (2) communicating informationregarding the mutations to the professional.

After the ratio of particular polypeptide markers, or presence ofparticular nucleic acid markers in a stool sample is reported, a medicalprofessional can take one or more actions that can affect patient care.For example, a medical professional can record the results in apatient's medical record. In some cases, a medical professional canrecord a diagnosis of an aero-digestive cancer, or otherwise transformthe patient's medical record, to reflect the patients medical condition.In some cases, a medical professional can review and evaluate a patientsentire medical record, and assess multiple treatment strategies, forclinical intervention of a patients condition. In some cases, a medicalprofessional can record a tumor site prediction with the reportedimitations. In some cases, a medical professional can request adetermination of ratio of particular polypeptide markers to predicttumor site. In some cases, a medical professional can review andevaluate a patients entire medical record and assess multiple treatmentstrategies, for clinical intervention of a patient's condition.

A medical professional can initiate or modify treatment of anaero-digestive cancer after receiving information regarding a ratio ofparticular polypeptide markers or the presence of nucleic acid markersin a patients stool sample. In some cases, a medical professional cancompare previous reports and the recently communicated ratio ofparticular polypeptide markers, or presence of nucleic acid markers, andrecommend a change in therapy. In some cases, a medical professional canenroll a patient in a clinical trial for novel therapeutic interventionof an aero-digestive cancer. In some cases, a medical professional canelect waiting to begin therapy until the patient's symptoms requireclinical intervention.

A medical professional can communicate the ratio of particularpolypeptide markers to a patient or a patients family. In some cases, amedical professional can provide a patient and/or a patients family withinformation regarding aerodigestive cancers, including treatmentoptions, prognosis, and referrals to specialists, e.g., oncologistsand/or radiologists. In some cases, a medical professional can provide acopy of a patient's medical records to communicate the ratio ofparticular polypeptide markers to a specialist.

A research professional can apply information regarding a subject'sratio of particular polypeptide markers to advance aerodigestive cancerresearch. For example, a researcher can compile data on the ratio ofparticular polypeptide markers, and/or presence of particular nucleicacid markers, with information regarding the efficacy of a drug fortreatment of aero-digestive cancer to identify an effective treatment.In some cases, a research professional can obtain a subject's ratio ofparticular polypeptide markers, and/or determine the presence ofparticular nucleic acid markers to evaluate a subject's enrollment, orcontinued participation in a research study or clinical trial. In somecases, a research professional can classify the severity of a subject'scondition, based on the ratio of particular polypeptide markers and/orthe levels of particular nucleic acid markers. In some cases, a researchprofessional can communicate a subject's ratio of particular polypeptidemarkers, and/or the presence of particular nucleic acid markers to amedical professional. In some cases, a research professional can refer asubject to a medical professional for clinical assessment of anaero-digestive cancer, and treatment of an aero-digestive cancer.

Any appropriate method can be used to communicate information to anotherperson (e.g., a professional). For example, information can be givendirectly or indirectly to a professional. For example, a laboratorytechnician can input the ratio of particular polypeptide markers and/orparticular nucleic acid markers into a computer-based record. In somecases, information is communicated by making a physical alteration tomedical or research records. For example, a medical professional canmake a permanent notation or flag a medical record for communicating adiagnosis to other medical professionals reviewing the record. Inaddition, any type of communication can he used to communicate theinformation. For example, mail, e-mail, telephone, and face-to-faceinteractions can be used. The information also can be communicated to aprofessional by making that information electronically available to theprofessional. For example, the information can be communicated to aprofessional by placing the information on a computer database such thatthe professional can access the information. In addition, theinformation can he communicated to a hospital, clinic, or researchfacility serving as an agent for the professional.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Multi-Marker Quantitation and a Q-LEAD Model

Most approaches at marker detection in stool have been qualitative. Whensuch qualitative approaches are applied to assay of multiple markers(targeting multiple markers is required with neoplasm detection duemolecular heterogeneity), sensitivity is achieved at the expense ofcompounded nor specificity. Non-specificity can lead to prohibitiveprogrammatic cost with population screening due to the expensive andunnecessary evaluations of false-positive tests. However, if markers arequantified, then a logistic model can be created to achieve both highsensitivity and high specificity. Such a logistic model can alsoincorporate population variables like gender and age to adjust cut.-offlevels for test positivity and thereby optimize assay performance in ascreening setting (FIG. 1). This Quantitative Logistic to EnhanceAccurate Detection (Q-LEAD) Model can be used with any marker class orcombination of markers as long as they can be quantified.

A combination of more than one marker was undertaken to achieve thedesired sensitivity and specificity for cancer detection. Binaryregression methods predicting disease as a function of diagnostic testsestimate the optimal combination of the teats for classifying a subjectas diseased or not. McIntosh and Pepe, Biometrics 58: 657-664 (2002). Alogistic regression model can assess the relationship between a binarydependent response variable such as presence or absence of disease andone or more independent predictor variables. The independent predictorsmay be qualitative (e.g., binary) or quantitative (e.g., a continuousendpoint). In the Q-LEAD model, the independent predictors can includesuch biological markers as K-ras, BMP3 and DNA concentration, andothers. Importantly, the model incorporates the demographic variables ofgender and age, as we have observed that both age and gender influencemolecular marker levels in stool. As average stool marker levelsincrease with age and male gender, failure to adjust for these variableswould yield suboptimal specificity in Men and elderly persons tested.Coefficients are estimated from the sample data for each term in themodel. The result of the model is a risk score for each subject. Cutoffsfor predicting disease state from this risk score can be determined inorder to maximize sensitivity and specificity of the marker combinationsfor predicting disease as desired. The inclusion of demographicvariables allows these cutoffs to be determined as a function of age andgender.

As an application of the Q-LEAD Model, the following was performed toevaluate a quantitative stool DNA assay approach targeting threeinformative markers for the detection of colorectal neoplasia. Subjectsincluded 34 with colorectal cancer, 20 with adenomas >1 cm, and 26 withnormal colonoscopy. Subjects added a DNA stabilization buffer with stoolcollection, and stools were frozen at −80° C. within 72 hours. Fromthawed stool aliquots, crude DNA was extracted by standard methods, andtarget genes were enriched by sequence capture. K-ras mutation score,methylation of BMP3 gene, and concentration of human DNA (245 bp length)were respectively quantified by a digital melt curve assay, real-timemethylation-specific PCR and real-time Alu PCR, respectively. Assayswere performed blinded. A logistic model, which incorporates threemarkers and gender, was constructed to analyze discrimination bycombined markers.

Age medians were 60 for patients with colorectal cancer, 66 for thosewith adenomas, and 61 for normal controls; and male/female distributionswere 23/11, 9/11, and 10/16, respectively. Detection rates of colorectalneoplasms were determined by individual quantitative markers atspecificity cut-offs of 96 percent and by combined markers (Table 1).Discrimination by combined markers was calculated using a qualitativebinomial method (each marker considered as positive or negative based onindividual 96 percent specificity) and by the Q-LEAD model (sensitivitydata shown at overall specificity of 96 percent).

TABLE 1 Specificity and sensitivity of cancer markers. SensitivitySpecificity Cancers Adenomas Both Individual Markers k-ras mutation 96%42% 32% 38% BMP3 methylation 96% 45% 32% 40% DNA concentration 96% 65%40% 56% Combined Markers Qualitative method 88% 90% 58% 78% Q-LEAD Model96% 90% 47% 76%

By quantitative assay and multivariable analysis of an informativemarker panel, stool DNA testing can achieve high sensitivity whilepreserving high specificity for detection of colorectal neoplasia. Theparticular three-marker combination of mutant K-ras, BMP3 methylation,and human DNA concentration represents a complementary, high-yieldpanel.

The above data set and additional data were analyzed as follows. Aquantitative stool DNA assay approach targeting four informative markersfor use in the detection of colorectal cancer and advanced adenoma wasevaluated. Subjects comprised 74 patients with colorectal cancer, 27with an adenoma >1 cm, and 100 with normal colonoscopy. Stools werecollected with a stabilization buffer before or >1 week aftercolonoscopy and were frozen at −80  C. within 24 hours of collection.From thawed stool aliquots, crude DNA was extracted as described above,and target genes were enriched by sequence-specific capture. Human DNAconcentration, K-ras and APC mutation scores, and BMP3 methylation weresensitively quantified by real-time Alu PCR, a digital melt curve assay(Zou et al., Gastroenterology, “High Detection Rates of ColorectalNeoplasia by Stool DNA fasting With a Novel Digital Melt Curve Assay,”(2008)), and real-time methylation-specific PCR, respectively. Assayswere performed blindly. Sensitivities and specificities of single makersand their combinations were analyzed.

Age medians were 61 for patients with colorectal cancer, 67 for thosewith adenomas, and 59 for normal controls; and, male/female ratios were52/22, 15/12, and 37/63, respectively. The table displays detectionrates of colorectal neoplasms by individual quantitative markers atspecificities of 90% and by combined markers at two specificities.(Table 2) Data in this table represent a training set and have not beenadjusted for age and gender. Yet, it is clear that the full panel of Mu,K-ras, APC, and BMP3 detected more neoplasms than any individual marker,p<0.05. At 90% specificity, the full panel detects more adenomas >3 cm(90%, 9/10) than <3 cm (47%, 8/17), p<0.05, and more colorectal cancersat stages (89%, 40/45) than at stages 1-11 (69%, 20/29) p<0.05. Neoplasmdetection rates were not affected by tumor location.

TABLE 2 Specificity and sensitivity of a four marker panel SensitivityColorectal cancers Adenomas Specificity I-II III-IV ≦3 cm >3 cmIndividual Markers APC mutation 90% 38% 40% 47% 50% k-ras mutation 90%46% 42% 24% 50% BMP3 methylation 90% 36% 38% 12% 30% DNA concentration90% 52% 76% 41% 50% Combined Markers 90% 69% 89% 47% 90%

In conclusion, a quantitative stool DNA assay system that incorporates astabilization buffer with specimen collection, high analyticalsensitivity, and a panel of broadly informative markers can achieve highdetection rates of both colorectal cancers and advanced adenoma.

Example 2 SITE Model and Matrix Marker Panel

A statistical model (Site of Tumor Estimate (SITE)) can be used topredict tumor site (e.g., anatomical location or tissue of origin) basedon inputs from sequencing data (such as by specific nucleic acid orcombination of nucleic acids mutated, specific mutational location on anucleic acid, and nature of mutation insertion, deletion, transition, ortransversion) or by any combination thereof) and/or data frompolypeptide or other types of markers.

A matrix marker panel was developed to include eight cancer-relatedgenes: K-ras, p53, APC, p16, EGFR, CTNNB1, BRAF, and SMAD4. The mutationfrequencies of these genes were tabulated against the six majoraero-digestive cancers based on literature or public database reviewsand on actual sequencing observations (Table 3). Literature frequencieswere derived from the COSMIC somatic mutation database, review articles,and texts.

TABLE 3 Matrix Panel of Markers by Tumor Site Total AD Cancer Site N p16p53 K-ras APC SMAD4 EGFR CTNNB1 BRAF Unique Literature Colorectal <5%50-75% 40% 85% 14% NA 13%  20% Pancreatic 85-100% 50-60% 80-90% 10-40%30% NA 3-8% <5% Lung 15-25%  25-75% 20-40%  5%  7% 30% 6% <5% Bile Duct15-60%  30-60% 40% 30-40% 17% NA 1% 14% Gastric 5-30% 20-50% 10% 20-60%NA <1% 30-50% <5% Esophageal 5-90% 40-90%  5-12%  5-60% NA NA <1%  <5%Actual (non-dbSNP) Colorectal 57  5% 47% 26% 75% 25% 12% 2% 30% 98%Pancreatic 24 29% 17% 62% 54%  8%  8% 4%  8% 83% Lung 56  9% 57%  9% 16%14% 14% 2%  4% 77% Bile Duct 15 13% 27% 13% 20% 13% 0  0    7% 67%Gastric 23 17% 22%  4% 35% 17%  4% 4% 0  65% Esophageal 24  4% 46%  4%33% 17%  4% 0    4% 79%

Some of the frequencies include other genetic alterations than simplysingle base changes and small insertions/deletions such as methylationevents, large homozygous deletions, and copy number changes. Suchalterations would not be reflected in the actual frequency table. Actualfrequencies were derived by sequencing coding and flanking gene regionsfrom 245 patient tissue samples reflecting the spectrum ofaero-digestive cancers. Only non-synonymous and splice site alterationsWere tabulated. When specific mutational hot-spot sites were able to beidentified for particular genes, only those sites were analyzed.

The matrix panel includes markers that are present to variable extentacross these tumors so that their aggregate use achieves high overallsensitivity and allows prediction of tumor site using the SITE Model.70% of tumors harbored one or more mutations from the eight gene panel.Some gene mutations, like those associated with p1.6, are common intumors above the colon but are rare for those in the colon. Mutant K-rasis frequent with colorectal and pancreatic cancers but infrequent in theother cancers. Mutations in EGFR clustered with lung and colorectaltumors and mutations in SMAD4 clustered with stomach and colorectaltumors. Genes such as p53, are commonly mutated across many differenttypes of cancers, but specific mutational locations or types ofmutations within p53 and other genes differ between tumor site (e.g.,Greenman et al., Nature, 446(7132):153-8 (2007); Soussi and Lozano,Biochem. Biophys. Res. Common., 331(3):834-42 (2005); Stephens et al.Nat. Genet., 37(6):590-2 (2005); Sjoblom et al., Science,314(5797):268-74 (2006); Wood et al., Science, 318(5853):1108-13 (2007);and Davies, Cancer Res., 65(17):7591-5(2005)) and can be factored in tothe SITE Model to predict tumor site. Single base substitutions were themost common type of mutation throughout the panel and those thatpredicted colorectal tumors included C-G and A-T transversions (FIG.12). Other tumor sites had similarly unique base change profiles. (Table4). Insertion/deletions mutations were most common with colorectaltumors, particularly adenomas.

TABLE 4 Specific Base Change Fractions in AD Tumors C > T T > C G > CA > T G > T T > G Tumor (G > A) (A > G) (C > G) (T > A) (C > A) (A > C)Head and Neck 0.38 0.12 0.38 0.12 Esophageal 0.8 0.07 0.13 Lung 0.3 0.110.02 0.13 0.34 0.09 Stomach 0.5 0.25 0.17 0.08 Pancreas 0.41 0.15 0.040.07 0.33 Bile Duct 0.5 0.12 0.25 0.12 CRA 0.34 0.05 0.11 0.11 0.34 0.03CRC 0.4 0.05 0.02 0.24 0.26 0.03

Polypeptide markers found in stool, such as by proteomic approaches, canalso be used to detect aero-digestive neoplasms and predict tumor site.The following was performed to identify and explore candidatepolypeptide markers in stool for the discriminate detection ofpancreatic cancer. Subjects included 16 cases with pancreatic cancer, 10disease controls (colorectal cancer), and 24 healthy controls. Wholestools were collected and frozen promptly in aliquots at −80° C. Thawedaliquots were centrifuged, and the aqueous supernatant from each wasanalyzed. Polypeptides were separated by 1-D electrophoresis, excisedfrom gels, and digested for mass spectrometric analysis using anLTQ-Orbitrap. Data outputs were searched using Mascot, Sequest, and X!Tandem programs against an updated Swissprot database that included allcataloged species. Unique peptide counts and ratio calculations wereperformed using Scaffold software.

Median age for pancreatic cancer cases was 67, for colorectal cancercontrols 63, and for healthy controls 62; and male/female distributionswere 9/7, 6/4, and. 9/15, respectively. Using shotgun-proteomictechniques on stools, two pancreatic enzymes (carboxypeptidases B andA2) were conspicuous, as unique spectral counts of the former werecommonly elevated with pancreatic cancer and of the latter commonlydecreased. Considered together as the ratio of carboxypeptidaseB/carboxypeptidase A2, pancreatic cancer cases were almost completelyseparated from colorectal cancer and healthy control groups. Medianratios were 0.9, 0.2, and 0.3, respectively. At a specificity cut-offfor the carboxypeptidase B/A2 ratio at 100% (i.e., ratios from normalcontrol and colorectal cancer stools all below cut-oft), sensitivity forpancreatic cancer was 86 percent (FIG. 2). Only two pancreatic cancerswere misclassified.

These results demonstrate that a stool assay of polypeptide markers canbe a feasible non-invasive approach to the detection pancreatic cancer.These results also demonstrate that multivariable analysis of specificpolypeptide ratios can be used.

In addition, poly peptide markers unique to colorectal neoplasms wereidentified (Table 5). For example, serotransferrin was found in stoolsfrom patients with colorectal cancer but not in those with pancreaticcancer. These markers when considered as part of a matrix panelcontribute both to overall sensitivity for tumor detection and helpdiscriminate colorectal from pancreatic cancer.

TABLE 5 Positive Stool Findings. Carboxypeptidase B/A2* SerotransferrinColorectal Cancer 0 60% Pancreatic Cancer 86% 0 Normal controls 0 0*ratio >0.75 considered positive

Another polypeptide in stool that is pancreatic cancer specific iselastase 3A. Methods and Results demonstrating this are as follows:

Stool Preparation

Samples were collected in phosphate buffered saline and either droppedoff in clinic or mailed in collection tub. Samples were homogenized andfrozen within 72 hours after receipt. Frozen stools were diluted 1:3 w:vin PBS (Roche, Cat#1666789). Diluted stools were stomached in a filterbag (Brinkman, BA6041/STR 177×305 mm) for 60 seconds on control settingand spun at 10,000 rpms for 30 minutes. Following an additional 10minute spin at 14,000 rpm, the supernatant was filtered through a0.45-μm syringe filter and analyzed. Total protein present in stool wasquantitated using a Bradford Protein Assay kit (Pierce).

1-Dimensional Electrophoresis

Stool supernatants were diluted 1:1 in Leammli-BME buffer and run on a10.5-14% gradient gel. Vertical slices were cut from 250 kDa to 15 kDaand in-gel digested using methods described elsewhere (e.g., Wilm etal., Nature, 379:466-469 (1996)). Bands were destained, dehydrated,digested in trypsin, extracted, and for MS analysis.

Mass Spectrometry

Lyophilized samples were reconstituted and injected with a flow of 500nL/min and a 75 minute gradient from 5-90% 98% acetonitrile. MS wasperformed in data dependent mode to switch automatically between MS andMS² acquisition on the three most abundant ions. Survey scans wereacquired with resolution r=60,000 at 40 m/z using FWHM with a targetaccumulation of 10⁶ counts. An isolation width of 2.5 m/z was applied.Exclusion mass width was 0.6 m/z on low end and 1.5 m/x on high end. Allacquisition and method development was performed using Xcaliher version2.0.

Database Searching All ms/ms

Samples were analyzed using Mascot (Matrix Science, London, UK; version2.1.03), Sequest (ThermoFinnigan, San Jose, Calif.; version 27, rev. 12)and X! Tandem (World Wide Web at “thegpm.org” version 2006.09.15.3).Mascot and X! Tandem were searched with a fragment ion mass tolerance of0.80 Da and a parent ion tolerance of 10.0 PPM. Sequest was searchedwith a fragment ion mass tolerance of 1.00 Da. Nitration of tyrosine wasspecified in Mascot as a variable modification.

Criteria for Polypeptide Identification

Scaffold (version Scaffold-01_06_06, Proteome Software Inc., Portland,Oreg.) was used to validate MS/MS based polypeptide identifications.Peptide identifications were accepted if they could he established atgreater than 95.0% probability as specified by the Peptide Prophetalgorithm (Keller et al, Anal. Chem., 74(20):5383-92 (2002)).Polypeptide identifications were accepted if they could be establishedat greater than 99.0 percent probability and contained at least twoidentified peptides. Polypeptide probabilities were assigned by theProtein Prophet algorithm (Nesvizhskii, Anal. Chem., 75(17):4646-58(2003)). Polypeptides that contained similar peptides and could not hedifferentiated based on MS/MS analysis alone were grouped to satisfy theprinciples of parsimony.

Specific to Elastase SA Ratio Determination

Ratios of elastase 3A were determined using spectral counts for eachpolypeptide. Ratios were determined by dividing the number of uniquepeptides of elastase 3A (determined using a composite ID from databasesearch modules Mascot, XTandem, and Seaquest and compiled in Scaffold)by the number of unique peptides from another polypeptide such aspancreatic alpha-amylase.

Results

The concentration of a specific pancreatic enzyme, elastase 3A, wasconsistently found to be elevated in the fecal supernatant of patientswith pancreatic cancer as compared to normal controls or patients withnon-pancreatic cancer (FIG. 3). These finding indicate that fecalconcentration of elastase 3A is an accurate marker for pancreaticcancer. In addition, the ratio of elastase 3A against other pancreaticenzymes (or other stable fecal polypeptides) was found to be especiallydiscriminant for pancreatic cancer and obviates the need to determineabsolute elastase 3A concentrations (FIG. 4). While mass spectrometrywas used to make these observations, elastase 3A levels and ratiosincluding elastase 3A can he measured using other methods as well.

Example Digital Melt Curve Assay for Scanning Mutations

A sensitive, rapid, and affordable method for scanning mutations inbodily fluids at high-throughput was developed. A melt curve assay is apost-PCR technique that can be used to scan for mutations in PCRamplicons. Mutations in PCR products can be detected by changes in theshape of the melting curve (heterozygote from mutant sample) compared toa reference sample (homozygote from wild-type sample) (FIG. 5). Meltcurve assay can scan all mutations in a DNA fragment <400 bp in lessthan 10 minutes, rather than individually targeting single mutations.Regular melt curve assays can detect mutations down to a limit of 5%mutant:wild-type and, thus, are not sensitive enough to detect mutationsin many biological samples. For instance, in stool, an analyticalsensitivity of 1% or less is required in order to detect precancerouspolyps or small early stage cancers. Importantly, a quantitative scorecan be given to density of target mutations (FIG. 6).

Digital PCR can augment the sensitivity of PCR to detect low abundancemutations. Gene copies can be diluted and distributed into 96 wells of aplate to increase the percentage of mutant copy to wild-type copies incertain wells. For example, if a stool DNA sample contains only 1% ofmutant BRAF copies compared to wild-type copies, distributing 300 copiesof BRAF gene into a 96-well plate can lead to three wells with anaverage mutant ratio of 33 percent (1:). After PCR amplification, thesethree wells with mutant copies can be detected by sequencing or otherapproaches. Since digital PCR requires PCR on a whole 96-well plate and96 sequencings (or other approaches) for each target, it can be slow andcostly.

The concept of digital melt curve assay is to combine the scanningability and speed of high resolution melt curve assay with thesensitivity of digital PCR. Miniaturizing and automating this technologydramatically lowers per assay cost and achieves high-throughputnecessary for population screening.

The following procedure was used to perform a digital melt curve assay.To prepare a DNA sample, gene target fragments (e.g., BRAF, K-ras, APC,p16, etc.) were captured from stool DNA using a sequence-specificcapture method and were quantified with real-time PCR. About 200 to 2000gene copies were mixed in tube with all PCR reagents. An average of 2 to20 copies (variable) were distributed to each well of a 96-well plate.PCR amplification was performed using specific primers on the plate(e.g., one target per plate). Final concentrations of PCR mastermix forDigital Melt Curve assays in a 96-well plate (500 μL dispersed to 96wells with each well containing 5 μL) were as follows: 2× pfxamplification buffer (Invitrogen), 0.3 mM each dNTP, 200 nM forwardprimer, 200 nM reverse primer, 1 mM MgSO₄, 0.02 unit/μL Platinum® pfxpolymerase (Invitrogen), and 0.1 unit/μL LcGreen+dye (Idaho Tech). Ahigh resolution melt curve assay was used to identify the wells withmutant copies. Sequencing was optionally performed to confirm 1 to 2representative wells.

In some cases, emulsion PCR can be used in place of digital PCR. In suchcases, each lipid drop can become a tiny PCR reactor of one singlemolecule of gene.

TABLE 6 Sequence Specific Capture Probes and DNA Primer Capture SEQ GeneTarget Region Probe/ Primer Oligo Sequence (5′→3′) ID No. KRASCondons 12/13 Probe GTGGACGAATATGATCCAACAATAGAGGTAAATCTTG 1Condons 12/13 Primer 1 AGGCCTGCTGAAAATGACTG 2 TTGTTGGATCATATTCGTCCAC 3Condons 12/13 Primer 2 TAAGGCCTGCTGAAAATGAC 4 ATCAAAGAATGGTCCTGCAC 5Condons 12/13 Primer 3 CGTCTGCAGTCAACTGGAATTT 6 TGTATCGTCAAGGCACTCTTGC 7Condons 12/13 Primer 4 CTTAAGCGTCGATGGAGGAG 8 TTGTTGGATCATATTCGTCCAC 3BRAF V600E Probe CCAGACAACTGTTCAAACTGATGGGACCCACTCCATC 9 V600E PrimerCCACAAAATGGATCCAGACA 10 TGCTTGCTCTGATAGGAAAATG 11 APC MCR Probe 1CAGATAGCCCTGGACAAACCATGCCACCAAGCAGAAG 12 MCR Probe 2TTCCAGCAGTGTCACAGCACCCTAGAACCAAATCCAG 13 MCR Probe 3ATGACAATGGGAATGAAACAGAATCAGAGCAGCCTAAAG 14 Condons 1286-1346 Primer 1TTCATTATCATCTTTGTCATCAGC 15 CGCTCCTGAAGAAAATTCAA 16 Condons 1346-1367Primer 2 TGCAGGGTTCTAGTTTATCTTCA 17 CTGGCAATCGAACGACTCTC 18Condons 1394-1480 Primer 3 CAGGAGACCCCACTCATGTT 19TGGCAAAATGTAATAAAGTATCAGC 20 Condons 1450-1489 Primer 4CATGCCACCAAGCAGAAGTA 21 CACTCAGGCTGGATGAACAA 22 Condon 1554 Primer 5GAGCCTCGATGAGCCATTTA 23 TCAATATCATCATCATCTGAATCATC 24 102457delCPrimer 6 GTGAACCATGCAGTGGAATG 25 ACTTCTCGCTTGGTTTGAGC 26 102457delCPrimer 7 CAGGAGACCCCACTCATGTT 19 CATGGTTTGTCCAGGGCTAT 27 102457delCPrimer 8 GTGAACCATGCAGTGGAATG 25 AGCATCTGGAAGAACCTGGA 28 TP53 Exon 4Probe AAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGA 29 Exon 4 PrimerCCCTTCCCAGAAAACCTACC 30 GCCAGGCATTGAAGTCTCAT 31 Exon 5 ProbeCATGGCCATCTACAAGCAGTCACAGCACATGACGGAG 32 Exon 5 PrimerCACTTGTGCCCTGACTTTCA 33 AACCAGCCCTGTCGTCTCT 34 Exon 6 ProbeAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGAC 35 Exon 6 PrimerCAGGCCTCTGATTCCTCACT 36 CTTAACCCCTCCTCCCAGAG 37 Exon 7 ProbeATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGA 38 Exon 7 PrimerCTTGGGCCTGTGTTATCTCC 39 GGGTCAGAGGCAAGCAGA 40 Exon 8 ProbeCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGC 41 Exon 8 PrimerGGGAGTAGATGGAGCCTGGT 42 GCTTCTTGTCCTGCTTGCTT 43

Example 4 Sensitive Detection of Mutations using a Digital Melt CurveAssay

The following was performed to develop a quantitative method forscanning gene mutations and to evaluate the sensitivity of thequantitative method for detecting target mutations in stool. A digitalmelt curve assay was designed by combining digital PCR to a modifiedmelt curve assay. Target genes in low concentration were PCR amplifiedwith a saturated DNA dye, LcGreen+, in a 96-well plate. Each wellcontained a small number of gene copies, which allowed highmutation/wild-type ratios in some wells that were then detected by meltcurve scanning using a LightScanner. Mutations were scored based on thenumber of wells containing mutant copies in a 96-well plate. To testsensitivity, mutant genes were spiked into a wild-type pool at 0.1, 0.5,1, 5, and 10% dilutions, and analyzed using digital melt curve assaywith 250-1000 gene copies per 96-well plate. This method was thenapplied in the stool detection of APC, p53, K-ras, and BRAF mutationsfrom 48 patients known to have mutations in one of these genes inmatched tumor tissue. Subjects included 9 patients with pancreaticcancer, 31 with colorectal cancer, and 8 with colorectal adenoma >1 cm.All mutations detected b digital melt curve were further confirmed bySanger sequencing.

The digital melt curve assay detected as few as 0.1% mutant copies foramplicons <350 bp using one 96-well plate (FIG. 7), compared to thedetection limit for regular melt curve of ≧5 percent. Each mutationscanning took 8-10 minutes with this manual approach. Mutations of APC,p53, K-ras, and BRAF genes were all successfully scanned with digitalmelt curve in quantitative fashion. Tissue-confirmed mutations weredetected from matched stools in 88 percent (42/48) of patients withgastrointestinal n.eopla.snis, including 89 percent with pancreaticcancer, 90 percent with colorectal cancer, and 75 percent withcolorectal adenoma >1 cm.

These results demonstrate that a digital melt curve assay can be ahighly sensitive approach for detecting mutations in stool, and that ithas potential for diagnostic application with both upper and lowergastrointestinal neoplasms.

Example 5 Using a Digital Melt Curve Assay to Detect Adenomas

Archived stools were used to evaluate a digital melt curve assay of DNAmarkers for detection of advanced adenomas and to compare the accuracyof the digital melt curve assay with that of occult blood testing and acommercial DNA marker assay method (EXACT Sciences). Average risksubjects collected stools without a preservative buffer and mailed themto central processing laboratories for banking and blinded stool testingby Hemoccult, HemoccultSENSA, and DNA marker assay. All subjectsunderwent a colonoscopy, and tissue from advanced adenomas was archived.Archival stools were selected from the 27 patients with a colorectaladenoma >1 cm found to harbor mutant K-ras on tissue analyses and fromthe first 25 age and gender matched subjects with normal colonoscopy.Standard methods were used to extract crude DNA from fecal aliquots, andK-ras gene was enriched by sequence capture. Mutations in the K-ras genewere quantified by a digital melt curve assay based on the number ofwells containing mutant gene copies in a 96-well plate and confirmed bysequencing.

Median age with adenomas was 67 and controls 71; and males/females were12./15 and 13/14, respectively. Median adenoma size was 1.5 cm (range1-3 cm). Based on a cut-off of >3 wells with mutant K-ras, the digitalmelt curve assay yielded an overall sensitivity of 59 percent foradenomas with a specificity of 92 percent; sensitivity for adenomas >2cm was 80 percent (8/10) and for those <2 cm was 47 percent (8/17),p=0.1. in these same stools, overall adenoma detection rates were 7percent by Hemoccult, 15 percent by HemoccultSENSA, and 26 percent bythe EXACT Sciences K-ras assay (p<0.05 for each vs. digital melt curve)(FIG. 8). Respective specificities were 92 percent, 92 percent, and 100percent.

These results demonstrate that an analytically-sensitive digital meltcurve assay method can be used to detect a majority of advancedcolorectal adenomas and improve yield over current stool testapproaches.

Example 6 Short DNA as a Cancer Marker

Free human DNA is present in all human stools and arises from cells shed(exfoliated) from the normal surface (mucosa) of the aero-digestivetract (mouth/throat, lungs, and all digestive organs) and from tumors orother lesions that may be present. It has been generally accepted that“long DNA” in stool reflects that presence of colorectal and otheraero-digestive tumors, in that cells exfoliated from cancers do notundergo typical cell death (apoptosis) which would shorten DNA.Specifically, because DNA from apoptotic cells would be broken down tofragment lengths shorter than 100 bp, long DNA was defined as beinglonger than 100 bp. Indeed, levels of long DNA were elevated in stoolsfrom patients with colorectal and other cancers as compared to thosefrom healthy controls (Zou et al., Cancer Epidemiol. Biomarkers Prev.15: 115 (2006); Ahlquist et al., Gastroenterology, 119:1219 (20W); andBoynton et al, Clin. Chem., 49:1058 (2003)). As such, long DNA in stoolcan serve as a marker for colorectal and other tumors.

“Short DNA” (i.e., <100 bp in length), however, was found to be as ormore discriminant than long DNA as a tumor marker in stool for detectionof both colorectal (FIG. 9) and pancreatic (FIG. 10) neoplasia.

Briefly, methods and materials similar to those described elsewhere wereused to detect short DNA present in stool samples (Zou et al., CancerEpidemiol. Biomarkers Prev., 15(6): 1115 (2006)). Total DNA wasextracted by isopropanol precipitation from 19 blinded stool samples: 9pancreatic adenocarcinoma, and 10 age/gender matched normals. The DNApellets were taken up in 8 mL of 10-fold diluted TE, pH 8. The Alusequence consists of conserved regions and variable regions. In theputative consensus Alu sequence, the conserved regions are the 25-bpspan between nucleotide positions 23 and 47 and the 16-bp span betweennucleotide positions 245 and 260. Although primers can be designed inany part of the Alu sequences, for more effectively amplifying Alusequences, the PCR primers are preferably completely or partially (atleast the 3′-regions of the primers) located in the conserved regions.Primers specific for the human Alu sequences were used to amplifyfragments of differing lengths inside Alu repeats. The sequences were asfollows:

Ampli- con size Primer Sequences 245 bpForward Primer: 5′-ACGCCTGTAATCCCAGCACTT-3′ (SEQ ID NO: 44)Reverse Primer: 5′-TCGCCCAGGCTGGAGTGCA-3′ (SEQ ID NO: 45) 130 bpForward Primer: 5′-TGGTGAAACCCCGTCTCTAC-3′ (SEQ ID NO: 46)Reverse Primer: 5′-CTCACTGCAACCTCCACCTC-3′ (SEQ ID NO: 47)  45 bpForward Primer: 5′-TGGTGAAACCCCGTCTCTAC-3′ (SEQ ID NO: 46)Reverse Primer: 5′-CGCccGGCTAATTTTTGTAT-3′ (SEQ ID No: 48)

Stool DNA was diluted 1:5 with Ix Tris-EDTA buffer (pH 7.5) for PCRamplification. Tris-EDTA buffer-diluted stool DNA (14) was amplified ina total volume of 25 μL containing Ix iQ SYBR Green Supermix (Bio-Rad,Hercules, Calif.), 200 nmol/L each primer under the followingconditions: 95° C. for 3 minutes followed by 40 cycles of 95° C., 60°C., and 72° C. for 30 seconds each. A standard curve was created foreach plate by amplifying 10-fold serially diluted human genomic DNAsamples (Novagen, Madison, Wis.). Melting curve analysis was made aftereach PCR to guarantee that only one product was amplified for allsamples.

Amplification was carried out in 96-well plates in an iCycler (Bio-Rad).Each plate consisted of stool DNA samples and multiple positive andnegative controls. Each assay was done in duplicate.

The following was performed to compare long DNA (245 bp) and short (45bp) human DNA in stool for detection of upper and lower GI neoplasms,and to assess the effect of GI tumor site on human DNA levels in stool.Subjects included 33 patients with colorectal cancer, 20 with colorectaladenomas >1 cm, 13 with pancreatic cancer, and 33colonoscopically-normal controls. Subjects added a preservative bufferto stools at time of collection to prevent post-defecation bacterialmetabolism of DNA, and stools were frozen within 8 hours at −80° C.Using a validated quantitative assay for human DNA (Zou et al.Epidemiol. Biomarkers Prev., 15:1115 (2006)), 245 bp and 45 bp Alusequences were amplified from all stools in blinded fashion.Sensitivities for long and short DNA were based on 97 percentspecificity cut-offs.

Age medians were 60, 66, 69, and 62 for colorectal cancer, colorectaladenoma, pancreatic cancer, and control groups, respectively, andmale/female distributions were 22/11, 9/11, 9/4, 11/21, respectively. Instools from neoplasm and control groups, amplification products werequantitatively greater for short DNA versus long DNA. Respectivesensitivities by long and short DNA were 66 percent and 62 percent withthe 29 distal colorectal neoplasms, 46 percent and 46 percent with the24 proximal colorectal neoplasms, and 15 percent and 31 percent (p=0.16)with the 13 pancreatic cancers. By Wilcoxan Rank-Stun test, effect ofneoplasm site on detection rates was significant for both long DNA(p=0.004) and short DNA (p=0.02). Among colorectal neoplasms, respectivesensitivities by long and short DNA were 48 percent and 52 percent withlesions <3 cm, 63 percent and 63 percent with those >3 cm, 64 percentand 61 percent with cancers, and 35 percent and 45 percent withadenomas.

These results demonstrate that short and long DNA can he comparablysensitive for stool detection of GI neoplasms. However, detection ratesvary with tumor site, being greatest with the most distal lesions andlowest with the most proximal ones. These results were consistent withsubstantial luminal degradation of DNA exfoliated from more proximal GIneoplasms.

It was also demonstrated that mutant gene markers in stool can bedetected to a greater extent if amplicon size is less than 70 bp,consistent with luminal degradation. Thus, short DNA can serve as amarker per se and as the target size for mutation detection.

Example 7 Use of Fecal Methylated BMP3 as a Neoplasia Marker

Stools from patients with colorectal tumors were found to containsignificantly elevated amounts of methylated BMP3 gene copies, but thosefrom normal individuals were found to contain none or only traceamounts. When fecal methylated BMP3 was assayed with an appropriateamplification method, colorectal cancers and premalignant adenomas werespecifically detected (FIG. 11). Fecal methylated BMP3 detected a higherpercentage of proximal colon tumors than distal tumors, so it can becombined with markers for distal colorectal tumors to createcomplementary marker panels. Fecal methylated BMP3 was very specificwith few false-positive reactions.

Similar results can be obtained using other genes and methods such asthose described elsewhere (Zou et al., Cancer Epidemiol. BiomarkersPrev., 16(12):2686 (2007)).

Example 8 Detecting Aero-Digestive Cancers by Stool DNA Testing

Tissue samples from patients with confirmed acre-digestive tumors wereextracted and sequenced to assess the presence or absence of somaticgene alterations. Germline DNA from the same patients were used ascontrols. Once an alteration was confirmed, a matched stool sample wastested for that alteration. Two separate methods were utilized to detectthe mutation in stool: Allele specific PCR and digital melt curveanalysis. For both methods, we focused on amplifying the shortestfragments possible (>100 bp) that have been shown to contain higherlevels of the mutant sequence.

Digital Melt Curve (DMC)

We studied 138 patients (69 cases with a GI neoplasm and 69age/sex-matched asymptomatic controls with normal colonoscopy) by first,identifying a mutation in neoplasm tissue, and then determining if thatspecific mutation could be detected in stool from that individual.Stools were collected with a stability butler and frozen at −80° C.until assayed.

Genes commonly mutated in GI neoplasms (TP 3, KRAS, APC, CDH1, CTNNB1,BRAF, SMAD4, and P16) were sequenced from DNA extracted from minortissue, to identify a target mutation for each case. Target genes wereisolated by hybrid capture (Table 7) and the tissue-confirmed somaticmutations were assayed in stool by the digital melt curve method, asdescribed in Example 1. Mutations detected in stool were confirmed bysequencing. Assays were performed blinded.

TABLE 7Sequence Specific Capture Probes and Primers for AD Cancer Mutation DetectionMUTATION IN TISSUE CAPTURE PROBE SEQ ID No. 12487C > CT: 167Q > Q/XATGGCCATCTACAAGCAGTCATAGCACATGACGGAGGTTGT 49 102447_102450het_delTGGTAGAGTGAACCATGCAGTGGAAAAGTGGCATTATAAGCCC 50 12410G > GA, 141C > C/YTTTGCCAACTGGCCAAGACCTACCCTGTGCAGCTGTG 51 102678het_delACAGATGCTGATACTTTATTACTTTTGCCACGGAAAGTACT 52 102594_102598het_delAGAGAAAAGCACCTACTGCTGAAAGAGAGTGGACCTAAGCAAG 53 102644_102646het_insGATGCTGCAGTTCAGAGGGGTCCAGGTTCTTCCAGATGC 54 102594_102595het_delAGTAAAGCACCTACTGCTGAAAAGAGAGAGTGGACCTAAGCAAG 55 102106het_delTCACAGGAAGCAGATTCTGCAATACCCTGCAAATAGCA 56 102442het_delTTTCAGAGTGAACCATGCAGGGAATGGTAAGTGGCATTAT 57 apc 102494C > CT: 1429Q > Q/XTCCAGATAGCCCTGGATAAACCATGCCACCAAG 58 apc 102557C > CT: 1450R > RACTCAAACAGCTCAAACCAAGTGAGAAGTACCTAAAAATAAA 59 apc 102140het_delAAGCAGAAATAAAAGAAAAGTTGGAACTAGGTCAGCTGA 60 apc 102494C > CT: 1429Q > Q/XTCCAGATAGCCCTGGATAAACCATGCCACCAAG 58 apc 102554het_delACTCAAACAGCTCAAACCAGCGAGAAGTACCTAAA 61 tp53 E5 12647A > AG: 193H > H/RTCTGGCCCCTCCTCAGCGTCTTATCCGAGTGGAAG 62 tp53 E5 12742G > GACCTATGAGCCGCCTGAGATCTGGTTTGCAACTGGG 63 tp53 E5 12706C > CT: 213R > R/XATGACAGAAACACTTTTTGACATAGTGTGGTGGTG 64 tp53 E4 12712A > AG: 215S > S/GGAAACACTTTTCGACATGGTGTGGTGGTGCCCTAT 65 tp53 E4 12388T > TC: 134F > F/LCTGCCCTCAACAAGATGCTTTGCCAACTGGCCAAG 66 tp53 E3 11606G > GA: 125T > T/TAAGTCTGTGACTTGCACAGTCAGTTGCCCTGAGGG 67 tp53 E6 13379C > CT: 248R > R/WGCATGGGCGGCATGAACTGGAGGCCCATCCTCACC 68 tp53 12E3 11326A >AC (splice site) TCTTTTCACCCATCTACCGTCCCCCTTGCCGTCCC 69 tp53 E6 13412G >GT: 259D > D/Y CCATCATCACACTGGAATACTCCAGGTCAGGAGCC 70 tp53 E4 12449G >GT: 154G > GN ACCCCCGCCCGTCACCCGCGTCC 71 tp53 E7 13872G > GT, 298E > E/XGGAACAGCTTTGAGGTGTGTGTTTGTGCCTGTCCT 72 APC 102843C > CG: 1545S > S/XTCAGAGCAGCCTAAAGAATGAAATGAAAACCAAGAGAAA 73 tp53 E4 12392G > GT, 135C >C/F CCTCAACAAGATGTTTTTCCAACTGGCCAAGACCT 74 APC 102557C > CT: 1450R > R/XCTCAAACAGCTCAAACCAAGTGAGAAGTACCTAAAAATAAA 59 tp53 E7 13819G > T: 280R >I CCTGTCCTGGGATAGACCGGCGCAC 75 tp53 13E4 11326A > AC (splice site)TCTTTTCACCCATCTACCGTCCCCCTTGCCGTCCC 69 tp53 E7 13412G > GT: 259D > D/YCCATCATCACACTGGAATACTCCAGGTCAGGAGCC 70 tp53 E5 12449g > GT: 154G > G/VACCCCCGCCCGTCACCCGCGTCC 71 tp53 E8 13872G > Gt: 298E > E/XGGAGCCTCACCACTAGCTGCCCCCAGG 76 tp53 E8 13813C > CG, 278P > P/RGTGTTTGTGCCTGTCGTGGGAGAGACCGGCG 77 tp53 E8 13851A > AT, 291K > K/XGGAAGAGAATCTCCGCTAGAAAGGGGAGCCTCA 78 smad4 E2 19049G > GA, 18118A > A/AGTTAAATATTGTCAGTATGCATTTGACTTAAAATGTGATAG 79 tp53 E8 13777G > GA: 266G >G/E TAGTGGTAATCTACTGGAACGGAACAGCTTTGAGGTG 80 tp53 E6 12653T > TC: 195I >I/T CCTCCTCAGCATCTTACCCGAGTGGAAGGAAAT 81 tp53 E7 13379C > CT: 248R > R/WGCATGGGCGGCATGAACTGGAGGCCCATCCTCACC 68 tp53 E6 12647A > AG: 193H > H/RTCTGGCCCCTCCTCAGCGTCTTATCCGAGTGGAAG 62 tp53 E6 12712A > AG: 215S > S/GGAAACACTTTTCGACATGGTGTGGTGGTGCCCTAT 65 tp53 E8 13872G > GT, 298E > E/XGGAGCCTCACCACTAGCTGCCCCCAGG 76 tp53 E7 13370G > GA: 245G > G/SAGTTCCTGCATGGGCAGCATGAACCGGAGGC 82 tp53 E4 11580het_delGCTGGGCTTCTTGCATTCTGGACAGCCAAGTCTGTGA 83 tp53 E5 12524A > AG, 179H > H/RTGCCCCCACCGTGAGCGCTGC 84 >tp53 E6 12661G > GT, 198E > E/XTCAGCATCTTATCCGAGTGTAAGGAAATTTGCGTGTGGA 85 tp53 E8 13872G > GT, 298E >E/X GGAGCCTCACCACTAGCTGCCCCCAGG 76 apc 102494C > CT: 1429Q > Q/XTCCAGATAGCCCTGGATAAACCATGCCACCAAG 58 apc 102557C > CT: 1450R > R/XCTCAAACAGCTCAAACCAAGTGAGAAGTACCTAAAAATAAA 59 apc 102140het_delAAGCAGAAATAAAAGAAAAGTTGGAACTAGGTCAGCTGA 60 apc 102494C > CT: 1429Q > Q/XTCCAGATAGCCCTGGATAAACCATGCCACCAAG 58 apc 102134G > GT: 1309E > E/XTGCAAATAGCAGAAATAAAATAAAAGATTGGAACTAGGTCA 86 apc102554het_delACTCAAACAGCTCAAACCAGCGAGAAGTACCTAAA 61 apc 102852het_insACTAAAGAATCAAATGAAAAACCAAGAGAAAGAGGCAGAA 87 Kras 5571G > GA: 12G > G/DGTGGTAGTTGGAGCTGATGGCGTAGGCAAGAGT 88 tp53 E4 12392G > GA: 135C > C/YCCTCAACAAGATGTTTTACCAACTGGCCAAGACCT 89 tp53 E5 12655C > CT: 196R > R/XCCTCCTCAGCATCTTATCTGAGTGGAAGGAAATTTGC 90 tp53 E6 13350G > GA: 238C > C/YTCCACTACAACTACATGTATAACAGTTCCTGCATGGG 91 tp53 E6 13420G > GACACTGGAAGACTCCAGATCAGGAGCCACTTGCC 92 tp53 E5 12712A > AG: 215S > S/GGAAACACTTTTCGACATGGTGTGGTGGTGCCCTAT 65 Kras 5571G > GA: 12G > G/DGTGGTAGTTGGAGCTGATGGCGTAGGCAAGAGT 88 P16 (ink4a) E1 19638A > ATGGAGAGGGGGAGTGCAGGCAGCGGG 93 P16 (ink4a) E2 23353G > GT: 447D > DYCCCAACTGCGCCTACCCCGCCACTC 94 P16 (ink4a) E1 19638A > ATGGAGAGGGGGAGTGCAGGCAGCGGG 93 p16(ink4a) E2 23402het_delT_GCCCGGGAGGGCTCCTGGACACGCTG 95 p16(ink4a) E2 23403C > CA: 484F > F/GCCCGGGAGGGCTTACTGGACACGCTGGT 96 ctnnbl 25541het_delTCAATGGGTCATATCACAGATTCTTTTTTTTAAATTAAAGTAACA 97 cdh 1 E9 76435het_delATCTTATCTCAAAAGAACAACAAAAAAGAGGAATCCTTTAG 98cdh1 E1 743_744het_insAGCCCTGCGCCCA GCGCCCAGCCCTGCGCCCATTCCTC 99cdh 1 E13 8685386854het_insA AAGTAAGTCCAGCTGGCAAAGTGACTCAGCCTTTGACTT 100cdh 1 E14 91472C > CT: 751N > N/NAGGATGACACCCGGGACAATGTTTATTACTATGATGAAG 101 cdh 1 E 15TTTTTTCTCCAAAGGACTGACGCTCGGCCTGAAGTG 102 92868_92896het delTTGACTTGAGCCAGCTGCACAGGGGCCTG cdh 1 E4 71669*het_delACAAGCAGAATTGCTCACTTTCCCAACTCCTCTCC 103 cdh 1 E7 74926G > GA: 289A > A/TGGTCACAGCCACAGACACGGACGATGATGTGAA 104 cdh 1 E1 736_742het_delTGCGCCCAGCCCTGCGCCCCTTCCTCTCCCG 105 p16(ink4a) E1 19638A > ATGGAGAGGGGGAGTGCAGGCAGCGGG 93 tp53 E4 12365A > AG: 126Y > Y/CTTCCTCTTCCTACAGTGCTCCCCTGCCCTCAAC 106 tp53 E4 12548G > GATGCTCAGATAGCGATGATGAGCAGCTGGGGCTG 107 p16(ink4a) E1 19810T > TG: 491I >I/S GGTCGGAGGCCGAGCCAGGTGGGTAGA 108 tp53 E7 13757G > GAGCTTCTCTTTTCCTATCCTAAGTAGTGGTAATCTACTGG 109 tp53 E7 13815G > GC: 279G >G/R TTGTGCCTGTCCTCGGAGAGACCGGCG 110 tp53 E7 13816G > GA: 279G > G/ETGTGCCTGTCCTGAGAGAGACCGGCGC 111 tp53 E5 12365A > AC, 126Y > Y/STTCCTCTTCCTACAGTCCTCCCCTGCCCTCAAC 112 tp53 E5 12491A > AT, 168H > HLTCTACAAGCAGTCACAGCTCATGACGGAGGTTGTGGA 113 113 113 kras 5570G > GC, 12G >G/R GTGGTAGTTGGAGCTGATGGCGTAGGCAAGAGT 88 tp53 E7 13370G > GA, 245G > G/SAGTTCCTGCATGGGCAGCATGAACCGGAGGC 82 apc 102864_102865het_delAGAAATGAAAACCAAGAGAAAGGCAGAAAAAACTATTGATTC 114 tp53 E5 12386T > TC, 133M >M/T CCTGCCCTCAACAAGACGTTTTGCCAACTGGCC 115 cdh1 E15 93059G > GAGCTCATCTCTAAGCTCAGGAAGAGTTGTGTCAAAAATGAGA 116 tp53 E8 13798G >GA: 273R > R/H CGGAACAGCTTTGAGGTGCATGTTTGTGCCTGTCCTGGG 117p53 E6, 12698_12701het_delAC (1 or 2TGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAG 118 AC repeats) P53 E8, 13824C >CT, 282R > R/W TGTCCTGGGAGAGACTGGCGCACAGAGGAAGAGAAT 119 APC 102151G >GA, 1314R > R/R AAGAAAAGATTGGAACTAGATCAGCTGAAGATCCTGTG 120P53 ES 12457 G > G/T CGCCCGGCACCCGCTTCCGCGCCATGGCCA 121 p53 E813812C >CG, 278P > P/A GTGTTTGTGCCTGTGCTGGGAGAGACCGGCG 122 APC 102686het_de1AAGGTTCTTCCAGATGCTGATACTTTATTACATTTTGC 123 APC het_delAG betweenGCGAGAAGTACCTAAAAATAAAGCACCTACTGCTGAA 124102594_102603 (1 of 5 AG repeats) APC 102240C > CA, 1344S > S/XCAGGGTTCTAGTTTATCTTAAGAATCAGCCAGGCACA 125 102676102680delACATTCCAGATGCTGATACTTTATTTTGCCACGGAAAGTACTC 126 12487C > CT: 167Q > Q/XATGGCCATCTACAAGCAGTCATAGCACATGACGGAGGTTGT 49 102447_102450het_delTGGTAGAGTGAACCATGCAGTGGAAAAGTGGCATTATAAGCCC 50 12410G > GA, 141C > C/YTTTGCCAACTGGCCAAGACCTACCCTGTGCAGCTGTG 51 102678het_delACAGATGCTGATACTTTATTACTTTTGCCACGGAAAGTACT 52 102594_102598het_delAGAGAAAAGCACCTACTGCTGAAAGAGAGTGGACCTAAGCAAG 127 102776A > AT: 1523R > R/XATGACAATGGGAATGAAACAGAATCAGAGCAGCCTAAAG 14 102644_102645het_insGATGCTGCAGTTCAGAGGGGTCCAGGTTCTTCCAGATGC 54 102594_102595het_delAGTAAAGCACCTACTGCTGAAAAGAGAGAGTGGACCTAAGCAAG 55 102106het_delTCACAGGAAGCAGATTCTGCAATACCCTGCAAATAGCA 56 102442het_delTTTCAGAGTGAACCATGCAGGGAATGGTAAGTGGCATTAT 57 apc 102494C > CT: 1429Q > Q/XTCCAGATAGCCCTGGATAAACCATGCCACCAAG 58 apc 102140het_delAAGCAGAAATAAAAGAAAAGTTGGAACTAGGTCAGCTGA 60 apc 102554het_delACTCAAACAGCTCAAACCAGCGAGAAGTACCTAAA 61 MUTATION IN TISSUESENSE PRIMER 1 (5′ TO 3′) SEQ ID No. 12487C > CT: 167Q > Q/XAGTACTCCCCTGCCCTCAAC 128 102447_102450het_delTGGT TTTGAGAGTCGTTCGATTGC129 12410G > GA, 141C > C/Y AGTACTCCCCTGCCCTCAAC 128 102678het_delATCCAGGTTCTTCCAGATGCT 130 102594_102598het_delAGAGA AGCTCAAACCAAGCGAGAAG131 102644_102646het_insG GGACCTAAGCAAGCTGCAGTA 132102594_102595het_delAG AGCTCAAACCAAGCGAGAAG 131 102106het_delTCAGACGACACAGGAAGCAGA 133 102442het_delT TTTGAGAGTCGTTCGATTGC 129apc 102494C > CT: 1429Q > Q/X GTGAACCATGCAGTGGAATG 25 apc 102557C >CT: 1450R > RA ACCACCTCCTCAAACAGCTC 134 apc 102140het_delACAGACGACACAGGAAGCAGA 133 apc 102494C > CT: 1429Q > Q/XGTGAACCATGCAGTGGAATG 25 apc 102554het_delA CATGCCACCAAGCAGAAGTA 21tp53 E5 12647A > AG: 193H > H/R CAGGCCTCTGATTCCTCACT 36 tp53 E5 12742G >GA CATAGTGTGGTGGTGCCCTA 135 tp53 E5 12706C > CT: 213R > R/XGTGGAAGGAAATTTGCGTGT 136 tp53 E4 12712A > AG: 215S > S/GGTGGAAGGAAATTTGCGTGT 136 tp53 E4 12388T > TC: 134F > F/LTGTTCACTTGTGCCCTGACT 137 tp53 E3 11606G > GA: 125T > T/TGTCTGGGCTTCTTGCATTCT 138 tp53 E6 13379C > CT: 248R > R/WTGGCTCTGACTGTACCACCA 139 tp53 12E3 11326A > AC (splice site)ACCTGGTCCTCTGACTGCTC 140 tp53 E6 13412G > GT: 259D > D/YCCTCACCATCATCACACTGG 141 tp53 E4 12449G > GT: 154G > GNGTGCAGCTGTGGGTTGATT 142 tp53 E7 13872G > GT, 298E > E/XGGAAGAGAATCTCCGCAAGA 143 APC 102843C > CG: 1545S > S/XATGCCTCCAGTTCAGGAAAA 144 tp53 E4 12392G > GT, 135C > C/FTGCCCTGACTTTCAACTCTGT 145 APC 102557C > CT: 1450R > R/XACCACCTCCTCAAACAGCTC 134 tp53 E7 13819G > T: 280R > ICTACTGGGACGGAACAGCTT 146 tp53 13E4 11326A > AC (splice site)ACCTGGTCCTCTGACTGCTC 140 tp53 E7 13412G > GT: 259D > D/YCCTCACCATCATCACACTGG 41 tp53 E5 12449g > GT: 154G > G/VGTGCAGCTGTGGGTTGATT 142 tp53 E8 13872G > Gt: 298E > E/XGGAAGAGAATCTCCGCAAGA 143 tp53 E8 13813C > CG, 278P > P/RCTACTGGGACGGAACAGCTT 146 tp53 E8 13851A > AT, 291K > K/XGCGCACAGAGGAAGAGAATC 147 smad4 E2 19049G > GA, 18118A > A/AAGGTGGCCTGATCTTCACAA 148 tp53 E8 13777G > GA: 266G > G/ETTTCCTTACTGCCTCTTGCTTC 149 tp53 E6 12653T > TC: 195I > I/TCAGGCCTCTGATTCCTCACT 36 tp53 E7 13379C > CT: 248R > R/WTGGCTCTGACTGTACCACCA 139 tp53 E6 12647A > AG: 193H > H/RCAGGCCTCTGATTCCTCACT 36 tp53 E6 12712A > AG: 215S > S/GGTGGAAGGAAATTTGCGTGT 136 tp53 E8 13872G > GT, 298E > E/XGGAAGAGAATCTCCGCAAGA 143 tp53 E7 13370G > GA: 245G > G/STGGCTCTGACTGTACCACCA 139 tp53 E4 11580het_delG CCCTTCCCAGAAAACCTACC 30tp53 E5 12524A > AG, 179H > H/R TGGCCATCTACAAGCAGTCA150 >tp53 E6 12661G > GT, 198E > E/X CAGGCCTCTGATTCCTCACT 36tp53 E8 13872G > GT, 298E > E/X GGAAGAGAATCTCCGCAAGA 143 apc 102494C >CT: 1429Q > Q/X CAGGAGACCCCACTCATGTT 19 apc 102557C > CT: 1450R > R/XCAGGAGACCCCACTCATGTT 19 apc 102140het_delA TTCATTATCATCTTTGTCATCAGC 15apc 102494C > CT: 1429Q > Q/X CAGGAGACCCCACTCATGTT 19 apc 102134G >GT: 1309E > E/X TTCATTATCATCTTTGTCATCAGC 15 apc102554het_delACAGGAGACCCCACTCATGTT 19 apc 102852het_insA GAGCCTCGATGAGCCATTTA 23Kras 5571G > GA: 12G > G/D AGGCCTGCTGAAAATGACTG 2 tp53 E4 12392G >GA: 135C > C/Y TGTTCACTTGTGCCCTGACT 137 tp53 E5 12655C > CT: 196R > R/XCAGGCCTCTGATTCCTCACT 36 tp53 E6 13350G > GA: 238C > C/YTGGCTCTGACTGTACCACCA 139 tp53 E6 13420G > GA CCTCACCATCATCACACTGG 141tp53 E5 12712A > AG: 215S > S/G GTGGAAGGAAATTTGCGTGT 136 Kras 5571G >GA: 12G > G/D AGGCCTGCTGAAAATGACTG 2 P16 (ink4a) E1 19638A > ATAGCCAGTCAGCCGAAGG 151 P16 (ink4a) E2 23353G > GT: 447D > DYCACCCTGGCTCTGACCAT 152 P16 (ink4a) E1 19638A > AT AGCCAGTCAGCCGAAGG 151p16(ink4a) E2 23402het_delT_ GACCCCGCCACTCTCAC 153p16(ink4a) E2 23403C > CA: 484F > F/ GACCCCGCCACTCTCAC 153ctnnbl 25541het_delT ATATTTCAATGGGTCATATCACAG 154 cdh 1 E9 76435het_delAGCCATGATCGCTCAAATACA 155 cdh1 E1 743_744het_insAGCCCTGCGCCCAACTTGCGAGGGACGCATT 156 cdh 1 E13 8685386854het_insA CATTCTGGGGATTCTTGGAG157 cdh 1 E14 91472C > CT: 751N > N/N CTGTTTCTTCGGAGGAGAGC 158cdh 1 E 15 TTCCTACTCTTCATTGTACTTCAACC 159 92868_92896het delTTGACTTGAGCCAGCTGCACAGGGGCCTG cdh 1 E4 71669*het_delA CGTTTCTGGAATCCAAGCAG 160cdh 1 E7 74926G > GA: 289A > A/T CCAGGAACCTCTGTGATGGA 161cdh 1 E1 736_742het_delTGCGCCC ACTTGCGAGGGACGCATT 156p16(ink4a) E1 19638A > AT AGCCAGTCAGCCGAAGG 151 tp53 E4 12365A >AG: 126Y > Y/C CACTTGTGCCCTGACTTTCA 33 tp53 E4 12548G > GACACATGACGGAGGTTGTGAG 162 p16(ink4a) E1 19810T > TG: 491I > I/STTCCAATTCCCCTGCAAA 163 tp53 E7 13757G > GA GGGACAGGTAGGACCTGATTT 164tp53 E7 13815G > GC: 279G > G/R CTACTGGGACGGAACAGCTT 146tp53 E7 13816G > GA: 279G > G/E CTACTGGGACGGAACAGCTT 146tp53 E5 12365A > AC, 126Y > Y/S CACTTGTGCCCTGACTTTCA 33 tp53 E5 12491A >AT, 168H > HL TGGCCATCTACAAGCAGTCA 150 TGGCCATCTACAAGCAGTCA 150TGGCCATCTACAAGCAGTCA 150 kras 5570G > GC, 12G > G/R AGGCCTGCTGAAAATGACTG2 tp53 E7 13370G > GA, 245G > G/S TGGCTCTGACTGTACCACCA 139apc 102864_102865het_delAG TGACAATGGGAATGAAACAGA 165 tp53 E5 12386T >TC, 133M > M/T TGTTCACTTGTGCCCTGACT 137 cdh1 E15 93059G > GACCAAAGCATGGCTCATCTCTA 205 tp53 E8 13798G > GA: 273R > R/HCTACTGGGACGGAACAGCTT 146 p53 E6, 12698_12701het_delAC (1 or 2GTGGAAGGAAATTTGCGTGT 136 AC repeats) P53 E8, 13824C > CT, 282R > R/WCTACTGGGACGGAACAGCTT 146 APC 102151G > GA, 1314R > R/RCAGACGACACAGGAAGCAGA 133 P53 ES 12457 G > G/T GTGCAGCTGTGGGTTGATT 142p53 E813812C > CG, 278P > P/A CTACTGGGACGGAACAGCTT 146APC 102686het_de1A CTGCAGTTCAGAGGGTCCAG 210 APC het_delAG betweenAGCTCAAACCAAGCGAGAAG 131 102594_102603 (1 of 5 AG repeats) APC 102240C >CA, 1344S > S/X CCCTAGAACCAAATCCAGCA 166 102676102680delACATTCTGCAGTTCAGAGGGTCCAG 167 12487C > CT: 167Q > Q/X AGTACTCCCCTGCCCTCAAC128 102447_102450het_delTGGT TTTGAGAGTCGTTCGATTGC 129 12410G >GA, 141C > C/Y AGTACTCCCCTGCCCTCAAC 128 102678het_delATCCAGGTTCTTCCAGATGCT 130 102594_102598het_delAGAGA AGCTCAAACCAAGCGAGAAG131 102776A > AT: 1523R > R/X TTTGCCACGGAAAGTACTCC 168102644_102645het_insG GGACCTAAGCAAGCTGCAGTA 132 102594_102595het_delAGAGCTCAAACCAAGCGAGAAG 131 102106het_delT CAGACGACACAGGAAGCAGA 133102442het_delT TTTGAGAGTCGTTCGATTGC 129 apc 102494C > CT: 1429Q > Q/XGTGAACCATGCAGTGGAATG 25 apc 102140het_delA CAGACGACACAGGAAGCAGA 133apc 102554het_delA CATGCCACCAAGCAGAAGTA 21 ANTISENSE PRIMER 2MUTATION IN TISSUE (5′ TO 3) SEQ ID No. 12487C > CT: 167Q > Q/XCTCACAACCTCCGTCATGTG 169 102447_102450het_delTGGT CATGGTTTGTCCAGGGCTAT27 12410G > GA, 141C > C/Y CTCCGTCATGTGCTGTGACT 170 102678het_delACACTCAGGCTGGATGAACAA 22 102594_102598het_delAGAGA AGCATCTGGAAGAACCTGGA28 102644_102646het_insG CACTCAGGCTGGATGAACAA 22 102594_102595het_delAGAGCATCTGGAAGAACCTGGA 28 102106het_delT TGCTGGATTTGGTTCTAGGG 171102442het_delT CATGGTTTGTCCAGGGCTAT 27 apc 102494C > CT: 1429Q > Q/XAGCTGTTTGAGGAGGTGGTG 172 apc 102557C > CT: 1450R > RAGCAGCTTGCTTAGGTCCACT 173 apc 102140het_delA TGCTGGATTTGGTTCTAGGG 171apc 102494C > CT: 1429Q > Q/X AGCTGTTTGAGGAGGTGGTG 172apc 102554het_delA GCAGCTTGCTTAGGTCCACT 173 tp53 E5 12647A > AG: 193H >H/R ACACGCAAATTTCCTTCCAC 174 tp53 E5 12742G > GA AACCACCCTTAACCCCTCCT175 tp53 E5 12706C > CT: 213R > R/X CAGTTGCAAACCAGACCTCA 176tp53 E4 12712A > AG: 215S > S/G CAGTTGCAAACCAGACCTCA 176tp53 E4 12388T > TC: 134F > F/L GCAGGTCTTGGCCAGTTG 177 tp53 E3 11606G >GA: 125T > T/T GCCAGGCATTGAAGTCTCAT 31 tp53 E6 13379C > CT: 248R > R/WCCAGTGTGATGATGGTGAGG 178 tp53 12E3 11326A > AC (splice site)GGGGACAGCATCAAATCATC 179 tp53 E6 13412G > GT: 259D > D/YGGGTCAGAGGCAAGCAGA 40 tp53 E4 12449G > GT: 154G > GNCTCCGTCATGTGCTGTGACT 170 tp53 E7 13872G > GT, 298E > E/XGCTTCTTGTCCTGCTTGCTT 43 APC 102843C > CG: 1545S > S/XTTTTTCTGCCTCTTTCTCTTGG 180 tp53 E4 12392G > GT, 135C > C/FCTGCACAGGGCAGGTCTT 181 APC 102557C > CT: 1450R > R/XGCAGCTTGCTTAGGTCCACT 173 tp53 E7 13819G > T: 280R > IGCGGAGATTCTCTTCCTCTG 182 tp53 13E4 11326A > AC (splice site)GGGGACAGCATCAAATCATC 179 tp53 E7 13412G > GT: 259D > D/YGGGTCAGAGGCAAGCAGA 40 tp53 E5 12449g > GT: 154G > G/VCTCCGTCATGTGCTGTGACT 170 tp53 E8 13872G > Gt: 298E > E/XGCTTCTTGTCCTGCTTGCTT 43 tp53 E8 13813C > CG, 278P > P/RGCGGAGATTCTCTTCCTCTG 182 tp53 E8 13851A > AT, 291K > K/XTTCTTGTCCTGCTTGCTTACC 183 smad4 E2 19049G > GA, 18118A > A/ATGGATTCACACAGACACTATCACA 184 tp53 E8 13777G > GA: 266G > G/ECACAAACACGCACCTCAAAG 185 tp53 E6 12653T > TC: 195I > I/TACACGCAAATTTCCTTCCAC 174 tp53 E7 13379C > CT: 248R > R/WCCAGTGTGATGATGGTGAGG 178 tp53 E6 12647A > AG: 193H > H/RACACGCAAATTTCCTTCCAC 174 tp53 E6 12712A > AG: 215S > S/GCAGTTGCAAACCAGACCTCA 76 tp53 E8 13872G > GT, 298E > E/XGCTTCTTGTCCTGCTTGCTT 43 tp53 E7 13370G > GA: 245G > G/SCCAGTGTGATGATGGTGAGG 178 tp53 E4 11580het_delG ACTGACCGTGCAAGTCACAG 186tp53 E5 12524A > AG, 179H > H/R CTGCTCACCATCGCTATCTG187 >tp53 E6 12661G > GT, 198E > E/X CCAAATACTCCACACGCAAA 188tp53 E8 13872G > GT, 298E > E/X GCTTCTTGTCCTGCTTGCTT 43 apc 102494C >CT: 1429Q > Q/X TGGCAAAATGTAATAAAGTATCAGC 20 apc 102557C > CT: 1450R >R/X TGGCAAAATGTAATAAAGTATCAGC 20 apc 102140het_delA CGCTCCTGAAGAAAATTCAA16 apc 102494C > CT: 1429Q > Q/X TGGCAAAATGTAATAAAGTATCAGC 20apc 102134G > GT: 1309E > E/X CGCTCCTGAAGAAAATTCAA 16 apc102554het_delATGGCAAAATGTAATAAAGTATCAGC 20 apc 102852het_insATCAATATCATCATCATCTGAATCATC 24 Kras 5571G > GA: 12G > G/DTTGTTGGATCATATTCGTCCAC 3 tp53 E4 12392G > GA: 135C > C/YGCAGGTCTTGGCCAGTTG 177 tp53 E5 12655C > CT: 196R > R/XACACGCAAATTTCCTTCCAC 174 tp53 E6 13350G > GA: 238C > C/YCCAGTGTGATGATGGTGAGG 178 tp53 E6 13420G > GA GGGTCAGAGGCAAGCAGA 40tp53 E5 12712A > AG: 215S > S/G CAGTTGCAAACCAGACCTCA 176 Kras 5571G >GA: 12G > G/D TTGTTGGATCATATTCGTCCAC 3 P16 (ink4a) E1 19638A > ATGAGGGGCTGGCTGGTC 189 P16 (ink4a) E2 23353G > GT: 447D > DYGGGTCGGGTGAGAGTGG 190 P16 (ink4a) E1 19638A > AT GAGGGGCTGGCTGGTC 189p16(ink4a) E2 23402het_delT_ CAGCTCCTCAGCCAGGTC 191p16(ink4a) E2 23403C > CA: 484F > F/ CAGCTCCTCAGCCAGGTC 191ctnnbl 25541het_delT TCAAATCAGCTATAAATACGAAACA 192cdh 1 E9 76435het_delA TCTCAGGGGGCTAAAGGATT 193cdh1 E1 743_744het_insAGCCCTGCGCCCA GAAGAAGGGAAGCGGTGAC 194cdh 1 E13 8685386854het_insA GGAAATAAACCTCCTCCATTTTT 195cdh 1 E14 91472C > CT: 751N > N/N CCGCCTCCTTCTTCATCATA 196 cdh 1 E 15TGCAACGTCGTTACGAGTCA 197 92868_92896het delTTGACTTGAGCCAGCTGCACAGGGGCCTG cdh 1 E4 71669*het_delA GCAGCTGATGGGAGGAATAA 198cdh 1 E7 74926G > GA: 289A > A/T TGAGGATGGTGTAAGCGATG 199cdh 1 E1 736_742het_delTGCGCCC GAAGAAGGGAAGCGGTGAC 194p16(ink4a) E1 19638A > AT CTCACAACCTCCGTCATGTG 169 tp53 E4 12365A >AG: 126Y > Y/C GCCAGTTGGCAAAACATCT 200 tp53 E4 12548G > GAAACCAGCCCTGTCGTCTCT 34 p16(ink4a) E1 19810T > TG: 491I > I/SCCCAACGCACCGAATAGT 201 tp53 E7 13757G > GA AGCTGTTCCGTCCCAGTAGA 202tp53 E7 13815G > GC: 279G > G/R GCGGAGATTCTCTTCCTCTG 182tp53 E7 13816G > GA: 279G > G/E GCGGAGATTCTCTTCCTCTG 182tp53 E5 12365A > AC, 126Y > Y/S GCCAGTTGGCAAAACATCT 200 tp53 E5 12491A >AT, 168H > HL CTGCTCACCATCGCTATCTG 187 TCACCATCGCTATCTGAGCA 203AACCAGCCCTGTCGTCTCT 34 kras 5570G > GC, 12G > G/R TTGTTGGATCATATTCGTCCAC3 tp53 E7 13370G > GA, 245G > G/S CCAGTGTGATGATGGTGAGG 178apc 102864_102865het_delAG GGTCCTTTTCAGAATCAATAGTTTT 204tp53 E5 12386T > TC, 133M > M/T GCAGGTCTTGGCCAGTTG 177 cdh1 E15 93059G >GA CTCAGGCAAGCTGAAAACAT 206 tp53 E8 13798G > GA: 273R > R/HGCGGAGATTCTCTTCCTCTG 182 p53 E6, 12698_12701het_delAC (1 or 2AGCTGTTTGAGGAGGTGGTG 172 AC repeats) P53 E8, 13824C > CT, 282R > R/WGCGGAGATTCTCTTCCTCTG 182 APC 102151G > GA, 1314R > R/RGTGACACTGCTGGAACTTCG 207 P53 ES 12457 G > G/T CTCCGTCATGTGCTGTGACT 170p53 E813812C > CG, 278P > P/A GCGGAGATTCTCTTCCTCTG 182APC 102686het_de1A CACTCAGGCTGGATGAACAA 22 APC het_delAG betweenAGCATCTGGAAGAACCTGGA 28 102594_102603 (1 of 5 AG repeats) APC 102240C >CA, 1344S > S/X TGTCTGAGCACCACTTTTGG 208 102676102680delACATTCACTCAGGCTGGATGAACAA 22 12487C > CT: 167Q > Q/X CTCACAACCTCCGTCATGTG 169102447_102450het_delTGGT CATGGTTTGTCCAGGGCTAT 27 12410G > GA, 141C > C/YCTCCGTCATGTGCTGTGACT 170 102678het_delA CACTCAGGCTGGATGAACAA 22102594_102598het_delAGAGA AGCATCTGGAAGAACCTGGA 28 102776A > AT: 1523R >R/X TTTCCTGAACTGGAGGCATT 209 102644_102645het_insG CACTCAGGCTGGATGAACAA22 102594_102595het_delAG AGCATCTGGAAGAACCTGGA 28 102106het_delTTGCTGGATTTGGTTCTAGGG 171 102442het_delT CATGGTTTGTCCAGGGCTAT 27apc 102494C > CT: 1429Q > Q/X AGCTGTTTGAGGAGGTGGTG 172apc 102140het_delA TGCTGGATTTGGTTCTAGGG 171 apc 102554het_delAGCAGCTTGCTTAGGTCCACT 173

Target imitations were not detected in control stools. Target mutationswere detected in stools from 68% (47/69) of patients with a GI neoplasm.Specifically, target mutations were detected in stools from 71% (36/51)of patients with cancer [40% (2/5) with oropharyngeal, 65% (11/17) withesophageal, 100% (4/4) with gastric, 55% (6/11) with pancreatic, 75%(3/4) with biliary or gallbladder, and 100% (10/10) with colorectal] andfrom 61% (11/18) with precancers [100% (2/2) with pancreaticintraductular papillary mucinous neoplasia and 56% (9/16) withcolorectal advanced adenoma]. Mutant copies in genes recovered fromstool averaged 0.4% (range 0.05-13.4%) for supracolonic and 1.4%(0.1-14.6%) for colorectal neoplasms, p=0.004 (Table 8).

TABLE 8 Digital Melt Curve Detection of Validated Mutations in AD CancerPatient Stool # ID Site Age Gender Tissue Mutation Stool DetectionMutation Frequency % Normal Control 1 1163 Head/Neck(pharynx) 73 M tp53YES 0.8 Neg 2 1250 Head/Neck(pharynx) 49 M tp53 NO Neg 3 1295Head/Neck(pharynx) 47 F tp53 NO Neg 4 1391 Head/Neck 65 M tp53 TP53 NO(both) Neg 5 1427 Head/Neck 60 M tp53 tp53 YES(p53-1), No (p53-2) 0.05Neg 1 745 Esophagus 84 F tp53 YES 0.4 Neg 2 769 Esophagus 56 F tp53 YES0.4 Neg 3 782 Esophagus 55 M tp53 NO Neg 4 789 Esophagus 61 M tp53 YES1.6 Neg 5 819 Esophagus 53 M tp53 YES 0.2 Neg 6 873 Esophagus 61 M tp53YES 0.2 Neg 7 906 Esophagus 55 M APC YES 0.8 Neg 8 1049 Esophagus 57 Mtp53 NO Neg 9 1064 Esophagus 72 F tp53 NO Neg 10 1067 Esophagus 72 Mtp53 YES 0.7 Neg 11 1103 Esophagus 78 M tp53 NO Neg 12 1199 Esophagus 66M tp53 YES 0.5 NEG 13 1307 Esophagus 51 M tp53 NO NEG 14 1373 Esophagus76 M tp53 YES 0.5 NEG 15 1414 Esophagus 66 M tp53 YES 0.1 NEG 16 1448Esophagus 82 M tp53 NO NEG 17 1072 Esophagus tp53 YES 0.4 NEG 1 798Stomach 81 M cdh1 YES 13.2 NEG 3 1221 Stomach 55 M cdh1 cdh1 YES(both)8,1.3 NEG 4 1224 Stomach 75 F smad4 cdh1 YES(smad4),No(CDH1) 0.2 NEG 51402 Stomach 56 M APC tp53 YES (p53) 0.1 NEG 1 848 Gall Bladder 67 Mtp53 YES 0.1 NEG 2 1315 Gall Bladder 57 F tp53 YES 1.4 NEG 1 1043 BileDuct 51 F APC NO NEG 2 1554 Bile Duct 77 M cdh1 YES 13.4 NEG 1 757Pancreatic Cancer in situ 78 M K-ras YES 0.2 NEG 2 1349 PancreaticCancer in situ 64 M K-ms YES 0.2 NEG 1 839 Pancreas 69 F tp53 YES 0.2NEG 2 1204 Pancreas 65 F p16 NO NEG 3 1253 Pancreas 63 F K-ras tp53Yes(k-ras), No(p53) 2 NEG 4 1400 Pancreas 71 F tp53 K-ras No (both) NEG5 1547 Pancreas 77 F tp53 NO NEG 6 1217 Pancreas K-ras NO NEG 7 1073Pancreas K-ras NO NEG 8 532 Pancreas K-ras YES 1 NEG 9 1592 PancreasK-ras P53 YES (both) 0.3 NEG 10 1695 Pancreas K-ras YES 0.2 NEG 11 1058Pancreas K-ras P53 APC YES(K-ras) 0.2 NEG 1 438 Colorectal Cancer 78 FAPC YES 1.2 NEG 2 446 Colorectal Cancer 74 M BRAF YES 0.4 NEG 3 529Colorectal Cancer 46 M K-RAS YES 1 NEG 4 489 Colorectal Cancer 73 MK-RAS YES 2.6 NEG 5 549 Colorectal Cancer 79 M BRAF YES 1.6 NEG 6 551Colorectal Cancer 69 M K-RAS YES 5.8 NEG 7 584 Colorectal Cancer 68 MK-RAS YES 1.4 NEG 8 894 Colorectal Cancer 57 M P53 APC YES(p53,APC)1.6,5 NEG 9 998 Colorectal Cancer 45 F APC KRAS YES(K-ras,APC) 0.6,0.8NEG 10 1009 Colorectal Adenoma 65 F P53 YES 12.9 NEG 1 513 ColorectalAdenoma 65 F APC YES 0.1 NEG 2 546 Colorectal Adenoma 61 M APC NO NEG 3547 Colorectal Adenoma 52 F APC NO NEG 4 568 Colorectal Adenoma 52 M APCYES 7.8 NEG 5 578 Colorectal Adenoma 71 F APC NO NEG 6 590 ColorectalAdenoma 54 F APC YES 3.2 NEG 7 701 Colorectal Adenoma 72 F APC NO NEG 8855 Colorectal Adenoma 75 M K-RAS YES 0.4 NEG 9 860 Colorectal Adenoma53 M APC YES 15.6 NEG 10 900 Colorectal Adenoma 64 F APC K-RAS No(both)NEG 11 962 Colorectal Adenoma 56 M K-RAS Yes 1 NEG 12 965 ColorectalAdenoma 82 M APC K-RAS No (both) NEG 13 991 Colorectal Adenoma 79 M APCK-RAS YES(K-ras), No(APC) 0.2 NEG 14 1135 Colorectal Adenoma 59 M K-RASYES 13 NEG 15 1231 Colorectal Adenoma 50 M APC NO NEG 16 1559 ColorectalAdenoma K-RAS YES 1 NEG

We also performed an initial pilot study with 10 stool samples frompatients with confirmed bile duct cancers to determine if DMC technologycould detect mutations in k-ras, a well characterized gene known bemutated in this population. K-ras mutations were detected in stools for3/10 or 4/10 bile duct cancers (depending on mutation score of 5 or 3,respectively) (Table 9). As K-ras is mutant in 30-40% of bile ductcancers, these results indicate that the detection assay is picking upthe appropriate proportion of cancer samples.

TABLE 9 K-ras mutation scores for patients with bile duct cancer. K-rasSample Mutation Mutation Detected # Pathology Score A B 520 BD Cancer 0528 BD Cancer 0 559 BD Cancer 0 558 BD Cancer 1 Codon 12 GAT 515 BDCancer 2 Codon 12 GAT 543 BD Cancer 2 Codon 13 GAC 806 BD Cancer 3 Codon12 TGT 539 BD Cancer 5 Codon 12 GAT Codon 13 GGA 512 BD Cancer 6 Codon12 GAT Codon 12 GAT 725 BD Cancer 25 Codon 13 GAC Codon 12 GAT; Codon 12GTT

Allele Specific PCR

The allele specific-PCR assay was a modified version of a previouslypublished method (e.g., Cha et al., Mismatch Amplification MutationAssay (MAMA): Application to the c-H-ras Gene PCR Methods andApplications, 2:14-20 (1992) Cold Spring Harbor Laboratory). TP53 genefragments were captured from stool DNA samples with probes specific tomutations identified in the matched tissue (Table 7). Copy numbers wereassessed by qPCR. Samples were adjusted to 10,000 fragments each andamplified with allele specific primer sets.

Sample F Primer R Primer A743 GACAGAAACACTTTAT CGGCTCATAGGG(SEQ ID No: 211) (SEQ ID No: 217) A848 ACACTTTTCGACAAG AAACCAGACCTCAG(SEQ ID No: 212) (SEQ ID No: 218) A789 CCTCAACAAGATAC CAGCTGCACAGG(SEQ ID No: 213) (SEQ ID No: 219) A782 GCCGCCTGAAA AGACCCCAGTTGC(SEQ ID No: 214) (SEQ ID No: 220) A873 GCGGCATGAAAT TTCCAGTGTGATGAT(SEQ ID No: 215) (SEQ ID No: 221) A769 CCCCTCCTCAGAG CTTCCACTCGGATAA(SEQ ID No: 216) (SEQ ID No: 222)The forward primer in each case is specific for each TP53 mutation.

Esophagus and Stomach

Targeting mutations found in esophageal cancers or those fromgastroesophageal junction (on p53, APC, or K-ras), the same mutation wasdetected by allele-specific PCR in matched stools from five of five(100%) cancers but in none of the controls (Table 10). The thresholdcycle (CO, designates the PCR cycle at which the product enters theexponential phase of amplification.

Gallbladder

Targeting a mutation confirmed in a gallbladder cancer, the samemutation was found in the matched stool from that patient usingallele-specific PCR (Table 10).

TABLE 10 Quantitative Mutant Allele Specific-PCR Results for MatchedAero-digestive Cancers Sample Gene # fragments Ct A769esophageal/gastric cancer p53 10K 71 N normal p53 10K >80 A782esophageal/gastric cancer p53 10K 38.8 N normal p53 10K 44.1 A745esophageal/gastric cancer p53 30K 42.5 N normal p53 30K 45.6 A873esophageal/gastric cancer p53 30K 37.8 N normal p53 30K 40.4 A848 gallbladder cancer p53 10K 22.4 N normal p53 10K 36.3 A789esophageal/gastric cancer p53 10K 25.9 N normal p53 10K 28.7

Example 9 Candidate Stool Polypeptide Markers Identified for ColorectalCancer and Precancerous Adenomas

The following list of polypeptides were identified by a statisticalanalysis model using all data generated from mass spectral of fecalprotein extracts: β2-macroglobulin, compliment C3 protein,serotransferrin, haptoglobin, carbonic anhydrase 1, xaa-pro dipeptidase,leukocyte elastase inhibitor, hemoglobin, glucose-6-phosphate, andcatalase. This list of polypeptides is in order of difference fromnormal. Thus, the mean spectral abundance for β2-macroglobulin is mostdifferent from normal for cancer and adenoma.

The statistical significance of relative poly peptide abundance betweennormal, adenoma, and colorectal cancer (CRC) was obtained usingnormalized spectral count data from a zero inflated poisson regressionmodel as an offset term in the protein specific differential expressionanalysis. The differential expression analysis also incorporated thezero inflated poisson regression model. Polypeptides were then rankedaccording to their statistical significance and whether the expressionprofile followed the clinically relevant pattern of Normal<Adenoma<CRC.Using a rule that a positive test required that any three of top sixmarkers be positive, the sensitivity and specificity of this panel wereboth 100% in a training set.

The listed polypeptides can he used individually or in any combinationto detect colorectal cancer or precancerous adenomas.

Example 10 Identification of Polypeptide Markers for Pancreatic Cancer

Potential polypeptide markers for pancreatic cancer prediction wereidentified. Utilizing a Scaffold (Proteome Software) side-by-sidecomparison of spectral abundances, ratios of the spectral counts ofcarboxypeptidase B (CBPB1_HUMAN) and Carboxypeptidase A1 (CBPA1_HUMAN)were compared. A value for carboxypeptidase B/A1 of 0.7 or higher waspredictive of pancreatic cancer (in normal stools, an average ratio of2:3 B/A1 was observed). Specificity for training data was 100% with asensitivity of 88%, while sensitivity from a validation set was 82% atthe same specificity.

Example 11 Use of Fecal Methylated ALX4 as a Neoplasia Marker

Stools from patients with colorectal tumors were found to containsignificantly elevated amounts of methylated ALX4 gene copies, but thosefrom normal individuals were found to contain none or only traceamounts. When fecal methylated ALX4 was assayed with an appropriateamplification method, colorectal cancers and premalignant adenomas werespecifically detected. At 90% specificity, fecal methylated ALX4detected 59% colorectal cancer and 54% premalignant adenomas, allowingfor the detection of both colorectal cancer and premalignara adenomas.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for characterizing a human biologicalsample comprising: (a) measuring a K-ras mutation score from a humanbiological sample; (b) measuring a methylation level of a CpG site ofBMP3 from the human biological sample; (c) determining whether or notthe human biological sample has an elevated K-ras mutation score and anelevated BMP3 methylation status as compared to a control from a subjectthat does not have premalignant or malignant colorectal cancer.
 2. Themethod of claim 1, wherein said measuring of said K-ras mutation scoreis measured by digital melt curve analysis.
 3. The method of claim 1,wherein said measuring of said K-ras mutation score is measured byquantitative allele-specific PCR.
 4. The method of claim 1, wherein thehuman biological sample is a stool sample.